Combinatorial libraries having aminodiol monomer subunits

ABSTRACT

Combinatorial libraries are constructed to include aminodiol monomer subunits connected by phosphodiester, phosphorothioate, or phosphoramidate linking moieties. Combinatorial libraries of the invention feature a plurality of functional groups attached to backbone and phosphoramidate combinatorial sites.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT application Ser. No.PCT/US95/00356 filed Jan. 11, 1995, which is a continuation-in-part ofU.S. application Ser. No. 08/180,134, filed Jan. 11, 1994; which is acontinuation in part of U.S. application Ser. No. 08/179,970, filed Jan.11, 1994. Each of these patent applications are assigned to the assigneeof this application and are incorporated by reference herein.

BACKGROUND OF THE INVENTION

Traditional processes of drug discovery involve the screening of complexfermentation broths and plant extracts for a desired biological activityor the chemical synthesis of many new compounds for evaluation aspotential drugs. The advantage of screening mixtures from biologicalsources is that a large number of compounds are screened simultaneously,in some cases leading to the discovery of novel and complex naturalproducts with activity that coul not have been predicted otherwise. Thedisadvantages are that many different samples must be screened andnumerous purifications must be carried out to identify the activecomponent, often present only in trace amounts. On the other hand,laboratory syntheses give unambiguous producte but the preparation ofeach new structure requires significant amounts of resources. Generally,the de novo design of active compounds based on the high resolutionstructures of enzymes has not been successful.

It is thus now widely appreciated that combinatorial libraries areuseful per se and that such libraries and compounds comprising them havegreat commercial importance. Indeed, a branch of chemistry has developedto exploit the many commercial aspects of combinatorial libraries.

In order to maximize the advantages of each classical approach, newstrategies for combinatorial deconvolution have been developedindependently by several groups. Selection techniques have been usedwith libraries of peptides (Geysen, H. M., Rodda, S. J., Mason, T. J.,Tribbick, G. and Schoofs, P. G., J. Immun. Meth. 1987, 102, 259-274;Houghten, R. A., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C.T. and Cuervo, J. H., Nature, 1991, 354, 84-86; Owens, R. A.,Gesellchen, P. D., Houchins, B. J. and DiMarchi, R. D., Biochem.Biophys. Res. Commun., 1991, 181, 402-408; Doyle, M. V., PCT WO94/28424; Brennan, T. M., PCT WO 94/27719); nucleic acids (Wyatt, J. R.,et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 1356-1360; Ecker, D. J.,Vickers, T. A., Hanecak, R., Driver, V. and Anderson, K., Nucleic AcidsRes., 1993, 21, 1853-1856); nonpeptides and small molecules (Simon, R.J., et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 9367-9371; Zuckermann,R. N., et al., J. Amer. Chem. Soc., 1992, 114, 10646-10647; Bartlett,Santi, Simon, PCT WO91/19735; Ohlmeyer, M. H., et al., Proc. Natl. Acad.Sci. USA, 1993, 90, 10922-10926; DeWitt, S. H., Kiely, J. S., Stankovic,C. J., Schroeder, M. C. Reynolds Cody, D. M. and Pavia, M. R., Proc.Natl. Acad. Sci. USA, 1993, 90, 6909-6913; Cody et al., U.S. Pat. No.5,324,483; Houghten et al., PCT WO 94/26775; Ellman, U.S. Pat. No.5,288,514; Still et al., PCT WO 94/08051; Kauffman et al., PCT WO94/24314; Carell, T., Wintner, D. A., Bashir-Hashemi, A. and Rebek, J.,Angew. Chem. Int. Ed. Engel., 1994, 33, 2059-2061; Carell, T., Wintner,D. A. and Rebek, J., Angew. Chem. Int. Ed. Engel., 1994, 33, 2061-2064;Lebl, et al., PCT WO 94/28028). A review of the above references revealsthat the most advanced of these techniques are those for selection ofpeptides and nucleic acids. Several groups are working on selection ofheterocycles such as benzodiazepines. With the exception of Rebek etal., scant attention has been given to combinatorial discovery of othertypes of molecules.

The majority of the techniques reported to date involve iterativesynthesis and screening of increasingly simplified subsets of oligomers.Monomers or sub-monomers that have been utilized include amino acids,amino acid-like molecules, i.e. carbamate precursors, and nucleotides,both of which are bifunctional. Utilizing these techniques, librarieshave been assayed for activity in either cell-based assays, or forbinding and/or inhibition of purified protein targets.

A technique, called SURF™ (Synthetic Unrandomization of RandomizedFragments), involves the synthesis of subsets of oligomers containing aknown residue at one fixed position and equimolar mixtures of residuesat all other positions. For a library of oligomers four residues longcontaining three monomers (A, B, C), three subsets each containing 27compounds would be synthesized (NNAN, NNBN, NNCN, where N representsequal incorporation of each of the three monomers). Each subset is thenscreened in a functional assay and the best subset is identified (e.g.NNAN). A second set of subsets is synthesized and screened, eachcontaining the fixed residue from the previous round, and second fixedresidue (e.g. ANAN, BNAN, CNAN, each containing 9 molecules). Throughsuccessive rounds of screening and synthesis, a unique sequence withactivity in the function assay can be identified. The SURF™ technique isdescribed in Ecker, D. J., Vickers, T. A., Hanecak, R., Driver, V. &Anderson, K., Nucleic Acids Res., 1993, 21, 1853-1856. The SURF™ methodis further described in PCT patent application WO 93/04204, the entiredisclosure of which is herein incorporated by reference.

The combinatorial chemical approach that has been most utilized to date,utilizes an oligomerization from a solid support using monomeric unitsand a defined connecting chemistry, i.e. a solid support monomerapproach. This approach has been utilized in the synthesis of librariesof peptides, peptoids, carbamates and vinylogous peptides connected byamide or carbamate linkages or nucleic acids connected by phosphatelinkages as exemplified by the citations in previous paragraphs above. Amixture of oligomers (pool or library) is obtained from the addition ofa mixture of activated monomers during the coupling step or from thecoupling of individual monomers with a portion of the support (beadsplitting) followed by remixing of the support and subsequent splittingfor the next coupling. In this monomeric approach, each monomeric unitwould carry a tethered letter, i.e., a functional group for interactionwith the target. Further coupling chemistry that allows for theinsertion of a tethered letter at a chemically activated intermediatestage is referred to as the sub-monomer approach.

The diversity of the oligomeric pool is represented by the inherentphysical properties of each monomer, the number of different monomersmixed at each coupling, the physical properties of the chemical bondsarising from the coupling chemistry (the backbone), the number ofcouplings (length of oligomer), and the interactions of the backbone andmonomer chemistries. Taken together, these interactions provide a uniqueconformation for each individual molecule.

There remains a need in the art for molecules which have fixedpreorganized geometry that matches that of targets such as proteins andenzymes, nucleic acids, lipids and other targets. The backbone of suchmolecules should be rigid with some flexibility, and such moleculesshould be easy to construct via automated synthesis on solid support

SUMMARY OF THE INVENTION

In accordance with this invention there are provided oligomericcompounds and libraries of such compounds comprising a plurality ofaminodiol monomer subunits joined by linking groups, wherein each ofsaid aminodiol monomer subunits have the Structure I, II, III, IV, V,VI, or VII;

wherein

R₁ is —T—L or a base labile protecting group;

T is a single bond, a methylene group or a group having formula:

—{[CR_(f)R_(g)]_(m)—B—[CR_(f)R_(g)]_(n)—[C(D)]_(p)—E—}_(q)—

 where:

D is ═O, =S, or =NR_(h);

B and E, independently, are a single bond, CH═CH, C≡C, O, S, NR_(h), orC₆-C₁₄ aryl;

each R_(f), R_(g) and R_(h) are independently H, alkyl or haloalkylhaving 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbonatoms, alkynyl having 2 to about 10 carbon atoms, or aryl having 7 toabout 14 carbon atoms;

m and n, independently, are 0 to 5;

p is 0 or 1;

q is 1 to about 10; and

L is H, substituted or unsubstituted C₂-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, substituted or unsubstituted C₄-C₇ carbocyclic alkyl,substituted or unsubstituted C₄-C₇ carbocyclic alkenyl, substituted orunsubstituted C₄-C₇ carbocyclic alkynyl, substituted or unsubstitutedC₆-C₁₄ aryl, an ether having 2 to 10 carbon atoms and 1 to 4 oxygen orsulfur atoms, a nitrogen containing heterocycle, a sulfur containingheterocycle, an oxygen containing heterocycle, a metal coordinationgroup, a conjugate group, halogen, hydroxyl (OH), thiol (SH), keto(C═O), carboxyl (COOH), amide (CONR), amidine (C(═NH)NRR), guanidine(NHC(═NH)NRR), glutamyl CH(NRR)(C(═O)OR), nitrate (ONO₂), nitro (NO₂),nitrile (CN), trifluoromethyl (CF₃), trifluoromethoxy (OCF₃), O-alkyl,S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino(NH₂), azido (N₃), hydrazino (NHNH₂), hydroxylamino (ONH₂), sulfoxide(SO), sulfone (SO₂), sulfide (S—), disulfide (S—S), silyl, a nucleosidicbase, an amino acid side chain, a carbohydrate, a biopharmaceuticallyactive moiety, or group capable of hydrogen bonding where thesubstituent groups are selected from hydroxyl, amino, alkoxy, alcohol,benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl,and alkynyl groups;

R₂ is hydrogen or C₁-C₁₀ alkyl;

R₃ and R₄ are independently hydrogen, an acid labile hydroxyl protectinggroup, a linking group or a conjugate group, wherein said linking grouphas the formula:

 wherein:

J₁ is ═O or ═S;

J₂ is OH or N(Y₀)T₀

Y₀ is H or [Q₂]_(j)—Z₂;

T₀ is [Q₁]_(k)—Z₁, or together Y₀ and T₀ are joined in a nitrogenheterocycle;

Q₁ and Q₂ independently are C₂-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₄-C₇ carbocylo alkyl C₄-C₇ carbocylo alkenyl, a heterocycle,an ether having 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms,a polyalkyl glycol, or C₇-C₁₄ aralkyl;

j and k independently are 0 or 1;

Z₁ and Z₂ independently are H, C₁-C₂ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, C₆-C₁₄ aryl, C₇-C₁₀ aralkyl, halogen, CH═O, OR_(a), SR_(b),NR_(c)R_(d), C(═NH)NR_(c)R_(d), CH(NR_(c)R_(d)), NHC(═NH)NR_(c)R_(d),CH(NH₂)C(═O)OH, C(═O)NR_(c)R_(d), C(═O)OR_(e), a metal coordinationgroup, a reporter group, a nitrogen-containing heterocycle, a purine, apyrimidine, a phosphate group, a polyether group, or a polyethyleneglycol group; and

provided that at least one of said aminodiol monomer subunits in saidoligomeric compound is not formula IV.

Further in accordance with this invention there a provided processes forpreparing oligomeric compounds and libraries of such compoundscomprising:

(a) selecting an aminodiol monomer subunit having the structure I, II,III, IV, V, VI, or VII:

 wherein

R₁ is a base labile amino protecting group;

R₂ is hydrogen or C₁-C₁₀ alkyl; and

one of R₃ or R₄ is hydrogen or an activated phosphite group and theother of R₃ or R₄ is an acid labile hydroxyl protecting group;

(b) attaching said aminodiol monomer subunit to a solid support to forma solid support bound aminodiol monomer subunit;

(c) treating said acid labile hydroxyl protecting group with a diluteacid to form a free hydroxyl group,

(d) reacting said free hydroxyl group with a further aminodiol monomersubunit having structure I, II, III, IV, V, VI, or VII, thereby formingan oligomeric compound bound to said solid support, said oligomericcompound containing a phosphite linkage;

(e) optionally iteratively repeating steps (c) and (d) to increase thelength of the oligomeric compound bound to said solid support;

(f) optionally, prior to step (c) or after step (d) oxidizing saidphosphite linkage to form a phosphate linking group wherein said linkinggroups are selected having formula:

 wherein:

J₁ is ═O or ═S;

J₂ is OH or N(Y₀)T₀

Y₀ is H or [Q₂]_(j)—Z₂;

T₀ is [Q₁]_(k)—Z₁, or together Y₀ and T₀ are joined in a nitrogenheterocycle;

Q₁ and Q₂ independently, are C₂-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₄-C₇ carbocylo alkyl or alkenyl, a heterocycle, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, apolyalkyl glycol, or C₇-C₁₄ aralkyl;

j and k independently, are 0 or 1;

Z₁ and Z₂, independently, are H, C₁-C₂ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, C₆-C₁₄ aryl, C₁-C₂ aralkyl, halogen, CH═O, OR_(a), SR_(b),NR_(c)R_(d), C(═NH)NR_(c)R_(d), CH(NR_(c)R_(d)), NHC(═NH)NR_(c)R_(d),CH(NH₂) C(═O)OH, C(═O)NR_(c)R_(d), C(═O)OR_(e), a metal coordinationgroup, a reporter group, a nitrogen-containinc heterocycle, a purine, apyrimidine, a phosphate group, a polyether group, or a polyethyleneglycol group; and

(g) prior to step (e) or after step (f) contacting said solid supportbound aminodiol monomer subunit or said support bound oligomericcompound with a base to remove said base labile amino protecting groupto form the solid suppoort bound aminodiol monomer subunit or supportbound oligomeric compound having a free amine, and derivatizing saidfree amine with a group of the formula —T—L; wherein:

T is a single bond, a methylene group or a group having formula:

—{[CR_(f)R_(g)]_(m)—B—[CR_(f)R_(g)]_(n)—[C(D)]_(p)—E—}_(q)—

 where:

D is ═O, ═S, or ═NR_(h);

B and E, independently, are a single bond, CH═CH, C≡C, O, S, NR_(h), orC₆-C₁₄ aryl;

each R_(f), R_(g) and R_(h) are, independently, H, alkyl or haloalkylhaving 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbonatoms, alkynyl having 2 to about 10 carbon atoms, or aryl having 7 toabout 14 carbon atoms;

m and n, independently, are 0 to 5;

p is 0 or 1;

q is 1 to about 10; and

L is H, substituted or unsubstituted C₂-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, substituted or unsubstituted C₄-C₇ carbocyclic alkyl,substituted or unsubstituted C₄-C₇ carbocyclic alkenyl, substituted orunsubstituted C₄-C₇ carbocyclic alkynyl, substituted or unsubstitutedC₁-C₁₄ aryl, an ether having 2 to 10 carbon atoms and 1 to 4 oxygen orsulfur atoms, a nitrogen containing heterocycle, a sulfur containingheterocycle, ar oxygen containing heterocycle, a metal coordinationgroup, conjugate group, halogen, hydroxyl (OH), thiol (SH), keto (C═O),carboxyl (COOH), amide (CONR), amidine (C(═NH)NRR), guanidine(NHC(═NH)NRR), glutamyl CH(NRR)(C(═O)OR), nitrat (ONO₂), nitro (NO₂),nitrile (CN), trifluoromethyl (CF₃), trifluoromethoxy (OCF₃), O-alkyl,S-alkyl, NH-alkyl, N-dialkyl O-aralkyl, S-aralkyl, NH-aralkyl, amino(NH₂), azido (N₃), hydrazino (NHNH₂), hydroxylamino (ONH₂), sulfoxide(SO), sulfone (SO₂), sulfide (S—), disulfide (S—S), silyl, a nucleosidicbase, an amino acid side chain, a carbohydrate, a biopharmaceuticallyactive moiety, or group capable of hydrogen bonding where thesubstituent groups are selected from hydroxyl, amino, alkoxy, alcohol,benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl,and alkynyl groups;

(h) optionally repeating steps (c) and (d) followed by step (g) toincrease the length of the oligomeric compound bound to said solidsupport;

(i) treating said oligomeric compound bound to said solid support withacid to deprotect any protecting groups; and

(j) cleaving said oligomeric compound from said solid support.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A and FIG. 1B are flow schemes illustrating certain processes ofthe inventions.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides combinatorial libraries of phosphatelinked aminodiol monomer subunits having phosphodiester,phosphorothioate, and phosphoramidate linkages. As used in the contextof the present invention an aminodiol monomer subunit (monomer subunitor backbone segment) is a cyclic or acyclic compound having a protectedreactive amino group either primary or secondary and two hydroxyls thatcan be either primary or secondary. One of the hydroxyls is protectedwith an acid labile protecting group and the other is in the form of thefree hrdroxyl or in the form of an H-phosphonate. The monomer subunitsof the invention are coupled using H-phosphonate chemistry to formoligomeric compounds which are substituted with diver functional groups.

Oligomeric compounds of the invention have reactive sites, also referredto as combinatorial sites, that may be combinatorialized with diversefunctional groups. Sites that are available for combinatorializinginclude reactive amino groups, H-phosphonate phosphorous groups that canbe oxidized with a) carbon tetrachloride and primary or secondary aminesto form a phosphoramidate, b) iodine and water to form phosphodiesters,c) sulfur in carbon disulfide to form phosphorothioates, and terminalhydroxyls. Terminal hydroxyls may also be substituted with a variety offunctional groups such as nucleotides or nucleosides to give a chimericcompound or may include many other groups such as conjugates, andreporter groups.

Functional groups may be attached directly to combina10 torial sites ormay include a tethering group to alter their orientation in space. Thefunctional groups are attached to the backbone segment andphosphoramidate moiety with or without intervening tethering groups.Tethering groups, as used in the context of this invention, are bivalentor polyvalent groups that have a primary or secondary amine or othersuitable group to react with an H phosphonate backbone segment of theinvention together with a second functional group capable of binding a“letter”. Such tethers can be used to position “letters” in space withrespect to the linear backbone of the oligomeric compound synthesized orto link letters that themselves do not include an amine group—necessaryto form a phosphoramidate linkage—as an inheren part of the letter. Aparticularly preferred group of compounds, when substituted with anappropriate amine functional group where necessary, useful as tetheringgroup include, but are not limited to C₂-C₁₀ alkyl, C₂-C₁₀ alkenylC₂-C₁₀ alkynyl, C₄-C₇ carboccylo-alkyl or alkenyl, heterocycles, anether having 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms,polyalkylene glycols and C₇-C₁₄ aralkyl groups. Other representativetethers useful in the practice of the invention are disclosed in U.S.application Ser. No. 08/116,801, filed Sep. 3, 1993, entitled“Thiol-Derivatized Nucleosides and Oligonucleosides” and U.S.application Ser. No. 117,363, filed Sep. 3, 1993, entitled“Amine-Derivatized Nucleosides and Oligonucleosides”, the disclosures ofwhich are hereby incorporated by reference.

The present invention provides for the addition of functional groupsonto the backbone of the oligomeric compounds on the solid support. Incontrast to previous methods, the synthesis of a large set of monomersubunits bearing various functional groups is not necessary. In oneaspect the oligomeric compounds of the invention are composed of threecomponents. The preparation of the combinatorial libraries begins withan aminodiol monomer subunit attached to the solid support directlythrough a linker stable to the synthesis conditions, but cleavable torelease the compound into solution at the end of the synthesis.Preferred linkers include esters, particularly succinic acid.Alternatively, the monomers can be coupled to a constant moiety attachedto the CPG, such as DMT ethylene glycol or a similar diol. Otherattachment points to the solid support are possible, for example:nucleosides, amino acids or other groups imparting pharmacokinetic,pharmacodynamic or other desirable properties.

The backbone segments are a structurally diverse set of aminodiols whichgive different relative orientations of the functional groups. Thebackbone segment amine function, also referred to as the reactive aminogroup, or amino combinatorial site, is protected with a base labileprotecting group and one hydroxyl is blocked with a protecting groupremovable with mild acid such as a dimethoxytrityl (DMT) ether. Thenitrogen protecting group is first removed from the backbone segment(s)and diverse functional groups added to the reactive amino group. Methodsfor achieving this are described below. A second monomer subunit canthen be added through H-phosphonate coupling after removal of the acidlabile protecting group on the terminal end of the compound on the solidsupport by for example, treating with dilute acid. The intermediatehydrogen phosphonate diester can be oxidized with, for example, asolution of CCl₄ Pyridine (1:1) containing 10% (v/v) of a primary orsecondary amine, resulting in the formation of a phosphoramidatelinkage. Alternatively the H-phosphonate linkage can be oxidized to thephosphodiester or the phosphorothioate using standard methods andtechniques. The second backbone segment can then be substituted withdiverse functional groups as above. Any combination of backbone segmentsand functional groups at the backbone segment amino combinatorial sitescan be introduced at any position of the oligomer and a large number ofamines can be used at the oxidation step. By repeating these syntheticsteps an oligomeric compound is synthesized having any desired sequenceof functional groups on the monomer subunits, and amidate derivatives atany linking position. Random positions are introduced into the libraryby dividing the solid support into portions before the addition of theappropriate reagent. Either the monomers, the monomer substituents orthe amines can be randomized. At least one position in the oligomersubsets must be fixed to allow iterative deconvolution. The librarysubsets are screened in the biological assay of interest. The mostactive subset defines the most active residue at the fixed position.Further rounds of synthesis and screening are used to determine thesequence of the most active compound in the library.

One feature of the present invention is the use of a nitrogen blockinggroup to block the reactive amino site. Once the first aminodiol monomersubunit is attached to the solid support, the nitrogen blocking groupcan be removed under basic (non hydrolytic) conditions. The nitrogen isthen derivatized with the diverse functional group of choice. This groupcan be attached to the amino combinatorial site via a variety of linkagegroups: amide, sulfonamide, carbamate, urea, aminoalkane, thiocarbamate,thiourea, etc. This can be accomplished by choosing the appropriateelectrophile to derivatize the nitrogen. For example, carboxylic acidscan be activated using peptide coupling reagents such as EDC, BOP orHATU. Other reagent which can be used include acid chlorides,-fluorides, -imidazolides, -anhydrides, sulfonyl chlorides,chloroformates, isocyanates, aldehydes (under reductive alkylationconditions), alkyl halides, isothiocyanates, etc. Thus each time afunctional group is desired in a library it is introduced via theappropriate coupling conditions using simple starting materials.

The aminodiol monomer subunits in the combinatorial library each bearfunctional groups e.g. “letters” in addition to those that formlinkages. When the aminodiol monomer subunits are linked together, thesefunctional groups provide diverse properties (“diversity”) to theresulting oligomeric compounds. The functional groups includehydrogen-bond donors and acceptors, ionic moieties, polar moieties,hydrophobic moieties, aromatic certers, and electron-donors andacceptors. Together, the properties of the individual monomerscontribute to the uniqueness of the oligomeric compounds in which theyare found. Thus, a library of such oligomers would have a myriad ofproperties i.e., “diversity.” Collectively, the properties of theindividual monomers that together form an oligomeric compound contributeto the uniqueness of such oligomeric compound and impart certaincharacteristics thereto for interaction with cellular, enzymatic ornucleic acid target sites.

A protecting group such as a member of the trityl fami preferably can beused as the acid labile protecting group of one of the two hydroxyls ofthe aminodiol monomer subunit. The trityl family includes at leasttrityl, monomethoxytrityl, dimethoxytrityl and trimethoxytrityl. Thedimethoxytrityl group is preferred and can be added by reacting theprimary hydroxyl group with 4,4′-dimethoxytrityl chloride. Otherhydroxyl protecting groups can be used. Representative hydroxylprotecting groups are described by Beaucage, et al., Tetrahedron 1992,48, 2223. Preferred hydroxyl protecting groups are acid-labile, such asthe trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl(MOX).

In other aspects of the present invention the use of acid labile groupswhich are stable to the trichloroacetic acid treatment used for DMTremoval such as BOC-type protecting groups are used. They are stable toextended TCA treatment, but are removed by trifluoroacetic acidsolutions (e.g. 5% in CH₂Cl₂). Another protecting group class which iscompatible to this methodology is the allyl class. These groups arecleaved using transition metal catalysts. These types of protectinggroup are particularly valuable in cases where the selectivedeprotection of a particular functional group is desired while theoligomer is still attached to the solid support, allowing a new reactivesite to be uncovered. Additional protecting group tactics are possible:e.g. photolabile protecting groups are also compatible with thismethodology.

In another aspect of the invention, nitrogen protecting groups that arestable to acid treatment and are selectively removed with base treatmentare used to make reactive amino groups selectively available forsubstitution. Examples of such groups are the FMOC (E. Atherton, R. C.Sheppard in The Peptides. S. Udenfriend, J. Meienhofer, Eds. AcademicPress, Orlando, 1987, vol 9, p1-38), and various substitutesulfonylethyl carbamates exemplified by the Nsc group (V. V. Samukov, A.N. Sabirov, P. I. Pozdnyakov, Tetrahedron Lett, 1994, 35, p7821; C. G.J. Verhart, G. I. Tesser, Rec. Trav. Chim. Pays-Bas, 1987, 107, p621).

Heterocycles, including nitrogen heterocycles, suitable for use asfunctional groups include, but are not limited to, imidazole, pyrrole,pyrazole, indole, 1H-indazole, a-carboline, carbazole, phenothiazine,phenoxazine, tetrazole, thiazole, oxazole, oxadiazole, benzoxazole,benzimidazole, triazole, pyrrolidine, piperidine, pyridine, quinoline,piperazine and morpholine groups.

Purines and pyrimidines suitable for use as functional groups includeadenine, guanine, cytosine, uridine, and thymine, as well as othersynthetic and natural nucleobase (nucleosidic bases) such as xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine,5-uracil (pseudo uracil), 4-thiouracil, 8-halo, amino, thiol, thioalkyl,hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine. Further purines and pyrimidines include those disclosedin U.S. Pat. No. 3,687,808, those disclosed in the Concise EncyclopediaOf Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I.,ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition 1991, 30, 613.

Alkyl, alkenyl, and alkynyl groups according to the invention includebut are not limited to substituted and unsubstituted straight chain,branch chain, and alicyclic hydrocarbons. Further, in the context ofthis invention, a straight chain compound means an open chain compound,such as an aliphatic compound, including alkyl, alkenyl, or alkynyl. Abranched compound, as used herein, comprises a straight chain compound,such as an alkyl, alkenyl, alkynyl compound, which has further straightor branched chains attached to the carbon atoms of the straight chain. Acyclic compound, as used herein, refers to closed chain compounds, i.e.a ring of carbon atoms, such as an alicyclic or aromatic compound. Thestraight, branched, or cyclic compounds may be internally interrupted,as in alkoxy or heterocyclic compounds. In the context of thisinvention, internally interrupted means that the carbon chains may beinterrupted with heteroatoms such as O, N, or S. However, if desired,the carbon chain may have no heteroatoms.

Further in the context of this specification aryl groups include but arenot limited to substituted and unsubstituted aromatic hydrocarbylgroups. Aralkyl groups include but are not limited to groups having botharyl and alkyl functionalities, such as benzyl and xylyl groups.Preferred aryl and aralkyl groups include, but are not, limited to,phenyl, benzyl, xylyl, naphthyl, tolyl, pyrenyl, anthracyl, azulyl,phenethyl, cinnamyl, benzhydryl, and mesityl. These can be substitutedor unsubstituted.

The aliphatic and aromatic groups as noted above may be substituted orunsubstituted. In the context of this invention, substituted orunsubstituted, means that the compounds may have any one of a variety ofsubstituents, in replacement, for example, of one or more hydrogen atomsin the compound, or may have no substituents. Typical substituents forsubstitution include, but are not limited to, hydroxyl, alkoxy, alcohol,benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, or alkyl, aryl,alkenyl, or alkynyl groups.

Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethers including polyethyleneglycols, and other moieties known in the art for enhancing thepharmacodynamic properties or the pharmacokinetic properties. Typicalconjugate groups include PEG groups, cholesterols, phospholipids,biotin, phenanthroline, phenazine, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes.

Metal coordination groups according to the invention include but are notlimited to hydroxamic acids, catecholamide, acetylacetone,2,2′-bipyridine, 1,10-phenanthroline, diacetic acid,pyridine-2-carboxamide, isoalkyldiamine, thiocarbamato, oxalate, glycyl,histidyl and terpyridyl. Other metal coordination groups are known, asfor example see Mellor, D. P., Chemistry of Chelation and ChelatingAgents in International Encyclopedia of Pharmacology and Therapeutics,Section 70, The Chelation of Heavy Metals, Levine, W. G. Ed., PergamonPress, Elmford, N.Y., 1979.

Solid supports according to the invention include controlled pore glass(CPG), oxalyl-controlled pore glass (see, e.g., Alul, et al., NucleicAcids Research 1991, 19, 1527), TentaGel Support—anaminopolyethyleneglycol derivatized support (see, e.g., Wright, et al.,Tetrahedro Letters 1993, 34, 3373) or Poros—a copolymer ofpolystyrene/divinylbenzene.

Non-reactive functionalities used as functional groups, such as groupsthat enhance pharmacodynamic properties, include groups that improveuptake and enhance resistance to enzymatic or chemical degradation.Non-reactive functionalities may also enhance pharmacokineticproperties. In the context of this invention, such groups improveuptake, distribution, metabolism or excretion. Non-reactivefunctionalities include, but are not limited to, alkyl chains,polyamines, ethylene glycols, steroids, polyamides, aminoalkyl chains,amphipathic moieties, and conjugate groups attached to any of thenitrogenous sites for attachment, as described above.

A number of functional groups can be introduced into compounds of theinvention in a blocked form and subsequently deblocked to form a final,desired compound. In general, a blocking group renders a chemicalfunctionality of a molecule inert to specific reaction conditions andcan later be removed from such functionality in a molecule withoutsubstantially damaging the remainder of the molecule (Green and Wuts,Protective Groups in Organic Synthesis, 2d edition, John Wiley & Sons,New York, 1991). For example, amino groups can be blocked as phthalimidogroups, as 9-fluorenylmethoxycarbonyl (FMOC) groups, and withtriphenylmethylsulfenyl, t-BOC or CBZ groups. Hydroxyl groups can beprotected as acetyl groups. Representative hydroxyl protecting groupsare described by Beaucage et al. Tetrahedron 1992, 48, 2223. Preferredhydroxyl protecting groups are acid-labile, such as the trityl,monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl(MOX). Chemical functional groups can also be “blocked” by includingthem in a precursor form. Thus, an azido group can be used considered asa “blocked” form of an amine since the azido group is easily convertedto the amine.

Additional functional groups according to the invention include but arenot limited to H, alkyl or substituted alkyl, alkenyl or substitutedalkenyl, alkynyl or substituted alkynyl, carbocyclic alkyl, alkenyl oralkynyl or substituted carbocyclic, or aryl or substituted aryl wherethe substituent groups are selected from hydroxyl, amino, alkoxy,alcohol, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl,alkenyl, or alkynyl groups; an ether having 2 to 10 carbon atoms and 1to 4 oxygen or sulfur atoms; a nitrogen, sulfur or oxygen containingheterocycle; a metal coordination group; a conjugate group; halogen;hydroxyl (OH); thiol (SH); keto (C═O); carboxyl (COOH); amide (CONR);amidine (C(═NH)NRR); guanidine (NHC(═NH)NRR); glutamyl CH(NRR)(C(═O)OR); nitrate

Functional groups of the invention can be represented by structure:

—T—L;

where T is a single bond, a methylene group or a group having formula:

—{[CR_(f)R_(g)]_(m)—B—[CR_(f)R_(g)]_(n)—[C(D)]_(p)—E—}_(q)—

 where:

D is ═O, ═S, or ═NR_(h);

B and E independently are a single bond, CH═CH, C≡C, O, S, NR_(h), orC₆-C₁₄ aryl;

each R_(f), R_(g) and R_(h) are independently H, alkyl or haloalkylhaving 1 to about 10 carbon atoms, alkenyl having 2 to about 10 carbonatoms, alkynyl having 2 to about 10 carbon atoms, or aryl having 7 toabout 14 carbon atoms;

m and n, independently, are 0 to 5;

p is 0 or 1;

q is 1 to about 10; and

L is H, substituted or unsubstituted C₂-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, substituted or unsubstituted C₄-C₇ carbocyclic alkyl,substituted or unsubstituted C₄-C₇ carbocyclic alkenyl, substituted orunsubstituted C₄-C₇ carbocyclic alkynyl, substituted or unsubstitutedC₆-C₁₄ aryl, an ether having 2 to 10 carbon atoms and 1 to 4 oxygen orsulfur atoms, a nitrogen containing heterocycle, a sulfur containingheterocycle, an oxygen containing heterocycle, a metal coordinationgroup, a conjugate group, halogen, hydroxyl (OH), thiol (SH), keto(C═O), carboxyl (COOH), amide (CONR), amidine (C(═NH)NRR), guanidine(NHC(═NH)NRR), glutamyl CH(NRR)(C(═O)OR), nitrate (ONO₂), nitro (NO₂),nitrile (CN), trifluoromethyl (CF₃), trifluoromethoxy (OCF₃), O-alkyl,S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino(NH₂), azido (N₃), hydrazino (NHNH₂), hydroxylamino (ONH₂), sulfoxide(SO), sulfone (SO₂), sulfide (S—), disulfide (S—S), silyl, a nucleosidicbase, an amino acid side chain, a carbchydrate, a biopharmaceuticallyactive moiety, or group capable of hydrogen bonding where thesubstituent groups are selected from hydroxyl, amino, alkoxy, alcohol,benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl,and alkynyl groups

As used in this specification, a “functional” group is one that, whenattached to a parent molecule, imparts to that molecule a particular andunique characteristic. It contributes diversity to the parent moleculeby rendering the parent molecule different in some way from what it wasbefore attachment of the group. Several chemical functional groups canbe attached to a particular molecule and when considered together, thesum total of their properties will impart global diversitycharacteristics to the parent molecule. Each set of combinations ofchemical functional groups on a particular molecule will modify theparent such that the parent molecule having each particular combinationsof groups will be different from the parent molecule having any of theother combinations of groups. When all of the combinations of the groupson the parent are considered, a library of compounds will be formed thatinclude all of the possible combinations of groups.

Oligomeric compounds of the invention can be synthesized with both theposition and the choice of the chemical functional groups predetermined,or allowed to be selected by combinatorial selection. In the context ofthis invention, “combinatorial” does not mean arbitrary, haphazard orindiscriminate. In the context of this invention, “combinatorial” isconstrued to mean that within the totality of the population ofoligomeric compounds that can be formed using a particular set offunctional groups and a particular location of combinatorial siteswithin the oligomeric compound, there will be sub-populations of each ofthe possible species. Thus, each of the different combinations of a)choice of functional group and b) positioning of the functional groupswill be represented.

“Combinatorial” is distinct from “random.” To illustrate thedistinction, if all or nearly all possible combinations are present inthe total molecular population, then it is a combinatorial population ofmolecules. If, however, only one or a small number of molecules fromthat total population is selected, then the selected molecule ormolecules might be randomly selected if it is picked at whim or willfrom the total population. When the totality of the population isconsidered, all species are present and it is not a random population.If a systematic selection was made until the totality of the populationwas exhausted, then all of the species would eventually be selected,however, the order of selection might be random. Thus, in certainpreferred embodiments, a pre-ordered selection and/or location ofchemical functional groups will be present. In further preferredembodiments, a combinatorialized population of all possible combinationsand ordering of the chemical functional groups is present. In evenfurther preferred embodiments, the sequence is modulated between fixedand combinatorial. This is especially useful, as for example, in certaindeconvolution strategies.

“Deconvolution” is construed to mean taking the totality of a populationand systematically working through that population to establish theidentity of a particular memeber, selected members, or all members ofthe population. In deconvoluting a combinatorial library of compounds,systematic selection is practiced until an individual oligomericcompound or a group of individual oligomeric compounds having aparticular characteristic, as for instance being an active species in aspecific functional assay, is identified.

Conjugate groups can also be used as functional groups in the presentinvention. Conjugate groups include intercalators, reporter molecules,polyamines, polyamides, polyethylene glycols, polyethers, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Typical conjugatesgroups include cholesterols, phospholipids, biotin, phenanthroline,phenazine, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamicproperties, in the context of this invention, include groups thatimprove oligomer uptake, enhance oligomer resistance to degradation,and/or strengthen sequence-specific hybridization with RNA or proteintargets. Groups that enhance the pharmacokinetic properties, in thecontext of this invention, include groups that improve compound uptake,distribution, metabolism or excretion. Representative conjugate groupsare disclosed in International Patent Application PCT/US92/09196, filedOct. 23, 1992, U.S. patent application Ser. No. 116,801, filed Sep. 3,1993, and U.S. Pat. No. 5,218,105. Each of the foregoing is commonlyassigned with this application. The entire disclosure of each isincorporated herein by reference.

Amines include amines of all of the above alkyl, alkenyl and aryl groupsincluding primary and secondary amines and “masked amines” such asphthalimide. Amines of this invention are also meant to includepolyalkylamino compounds and aminoalkylamines such as aminopropylaminesand further heterocycloalkylamines, such as imidazol-1, 2, or4-yl-propylamine.

Amino groups amenable to the present invention have the formula N(Y₀)T₀,wherein:

Y₀ is H, or [Q₂]_(j)—Z₂;

T₀ is [Q₁]_(k)—Z₁, or together Y₀ and T₀ are joined in a nitrogenheterocycle;

Q₁ and Q₂ independently, are C₂-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₄-C₇ carbocylo alkyl or alkenyl, a heterocycle, an etherhaving 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, apolyalkyl glycol, or C₇-C₁₄ aralkyl;

j and k independently, are 0 or 1;

Z₁ and Z₂, independently, are H, C₁-C₂ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, C₆-C₁₄ aryl, C₇-C₁₅ aralkyl, a halogen, CH═O, OR_(a), SR_(b),NR_(c)R_(d), C(═NH)NR_(c)R_(d), CH(NR_(c)R_(d)), NHC(═NH)NR_(c)R_(d),CH(NH₂)C(═O)OH, C(═O)NR_(c)R_(d), C(═O)OR_(e), a metal coordinationgroup, a reporter group, a nitrogen-containing heterocycle, a purine, apyrimidine, a phosphate group, a polyether group, or a polyethyleneglycol group.

To synthesize a combinatorial library having a large degree of chemicaldiversity is an important aspect of the present invention. Chemicaldiversity is introduced at one level by varying the nature of thephosphorus linkage. Phosphorus linkages amenable to the presentinvention include phosphodiester (OPO), phosphorothioate (OPS), andphosphoramidate (OPN). The desired combinatorial library can be preparedwith a single type of phosphorus linkage, or with different linkages ateach position of the oligomer. For example, a single OPS linkage can beselectivey introduced at any position in a OPO oligomer. In fact, allpossible combinations of OPO, OPS, and OPN linkages can be introducedselectively into the oligomeric compounds. The presence or absence of atype of linkage at a particular position in an oligomer will haveprofound effects on the properties of the molecule.

In the case of phosphoramidate linked libraries, a further level ofdiversity is possible by oxidizing the H-hosphonate diester linkage witha solution such as CCl₄ Pyridine (1:1) containing 10% (v/v) of a primaryor secondary amine, resulting in the formation of a phosphoramidatelinkage. Any of the H-phosphonate monomer subunits can be introduced atany position of the oligomer and a large number of amines can be used atthe oxidation step. Thus it is possible to introduce a wide variety ofamines into the oligomeric compound at H-phosphonate linkages byoxidation to the OPN linkage. It is possible to have the same aminesubstituents at each OPN linkage or a different amine at each position.In the preparation of a combinatorial library from a set of monomersubunits, amines and function groups, all possible combinations aresynthesized simultaneously.

Chemical diversity can be generated at several levels in SURF™libraries. We have described below the preparation of a number ofmonomer subunits. These monomers have been prepared to explore twoaspects of chemical diversity: first a wide number of functional groupsare available, covering a range of chemical properties. Second, thesefunctional groups can be attached directly to combinatorial sites or viatethering groups to combinatorial sites. Many different tethering groupsare commercially available and are amenable to the present invention.The use of tethering groups alters the presentation of functionalgroups, in space in different ways, allowing variable flexibility.

Hydrogen phosphonate chemistry allows additional chemical modificationsto be introduced into oligomeric compounds. Oligonucleotidephosphodiesters and phosphorothioates have been prepared using thisapproach,(see Froehler, B. C., Matteucci, M. D. Tetrahedron Lett. 1986,27, 469-472), as well as oligonucleotide phosphoramidates (see Froehler,B. C. Tetrahedron Lett. 1986, 27, 5575-5579. Letsinger, R. L., Singman,C. N., Histand, G., Salunkhe, M. J. Am. Chem. Soc. 1988, 110, 4470-4471.The synthesis of oligomeric compounds containing both phosphodiestersand phosphoramidates was reported, as well as the use of phosphoramiditechemistry in conjunction with the synthesis of phosphoramidates (seeJung, P. M., Histand, G., Letsinger, R. L. Nucleosides & Nucleotides,1994, 13, 1597-1605). In this latter work, alternating phosphodiesterand phosphoramidate oligomeric compounds were prepared by couplingphosphoramidites and H-Phosphonates to the growing oligomer, followed bythe appropriate oxidation step. In general, however, all the examplesdescribed thus far have incorporated the same amine substitution at allphosphoramidate linkages in the oligomer. These studies have shown thefeasibility of using the phosphoramidate bond as an additional site forthe incorporation of diverse functional groups.

A wide variety of amines can be used in the oxidative step, and themonomers of the present invention support the necessary chemistry. Thus,for the preparation of combinatorial libraries incorporatingphosphoramidate linkages, the monomer subunits of the present inventionused as the corresponding H-Phosphonate monoesters. In one aspect of thepresent invention this is accomplished using PCl₃ and imidazole as thephosphitylating reagent (see Garegg, P. J., Regberg, T., Stawinski, J.,Strömberg, R. Chem. Scr. 1986, 26, 59-62). These H-phosphonates monomersubunits may be oligomerized on solid support by activation withpivaloyl chloride, adamantoyl chloride or other appropriate activatingagent. The intermediate H-Phosphonate diesters are oxidized to thephosphate diesters in high yields using iodine in aqueous pyridine. Thisallowed for the comparison of the coupling efficiency of theH-phosphonate and phosphoramidite methods. Essentially the same couplingefficiency is achieved with both methodologies. The H-phosphonatediesters are converted to phosphoramidates by the use of a 10% solutionof the appropriate amine in pyridine/CCl₄ (1:1). Under these conditions,a H-phosphonate diester is oxidized to a phosphoryl chloride via anArbuzov reaction, followed by displacement of the chloride by a primaryor secondary amine. The second step has proven to be quite general, witha wide variety of amines giving satisfactory yields. Moreover, the yieldof phosphoramidate is comparable to the yield of phosphodiester.

Several types of libraries are available through this methodology. Thesimplest kind is a library made from a set of monomer subunits of thepresent invention, and a set of 4 to 16 or more functional groups, of 2to 10 or more monomer subunits in length, which is substituted atphosphorus with a single amine. These libraries are prepared by splitbead synthesis, following the H-phosphonate synthesis protocol ratherthan phosphoramidite chemistry.

In one aspect of the present invention, intermediate H-phosphonatediesters are left intact until the final step. At that point theoligomer library pools are oxidized with CCl₄/Pyridine containing 10% ofthe appropriate primary or secondary amine. The library therefore iscomposed. of all possible sequences of the monomers, separated intosubsets unique at a fixed position, linked together by a constantphosphoramidate linkage. It should be evident that. the final propertiesof the library will be determined by the choice of amine used in theoxidation step. Thus, water solubility, pharmacokinetics andpharmacodynamics of the library components can be modulated by thechoice of amine.

It is also possible to prepare oligomer libraries with mixed linkages byhaving an intermediate oxidation step (see Gryaznov, S. M., Sokolova, N.I. Tetrahedron Lett. 1990, 31, 3205-3208; Gryaznov, S. M., Potapov, V.K. Tetrahedron Lett. 1991, 32, 3715-3718; Farooqui, F., Sarin, P. S.,Sun, D., Letsinger, R. L. Bioconjugate Chem., 1991, 2, 422-426; Iso, Y.,Yoneda, F., Ikeda, H., Tanaka, K., Fuji, K. Tetrahedron Lett. 1992, 33,503-506). Thus, a portion of the oligomer library is synthesized byH-phosphonate chemistry, which can be oxidized with (R₂NH, CCl₄/Py orS8, CS₂/TEA or H₂O, CCl₄/Py), and a second portion of the librarysynthesized and oxidized with a second set of reagents. This creates achimeric library, where a segment of the random oligomeric compounds ineach subset has a different linkage than the rest of the molecule.

By extension of this methodology, it is possible to incorporate adifferent linkage at each position of the oligomer library by having adifferent oxidation step after each monomer subunit coupling. Thelinkage can be combinatorialized by performing a separate oxidation on aportion of the H-phosphonate diester-linked solid support, followed bypooling of the subsets in the same way that the monomer subunitpositions are randomized. Thus, each monomer and the linkage betweenthem can be randomized by a split synthesis strategy.

One advantage of the present invention is that the simple design ofmonomer subunits of the inventions allows for combining rational drugdesign with screen mechanisms for thousands of compounds. This isachieved by uEing the monomer subunits of the invention in acombinatorial techniques such as the SURF™ strategies.

In one preferred embodiment, functional groups appended to oligomericcompounds of the invention are selected for their potential to interactwith, and preferably inhibit, the enzyme PLA₂. Thus, the oligomericcompounds of the invention can be used for topical and/or systematictreatment of inflammatory diseases including atopic dermatitis andinflammatory bowel disease. In selecting the functional groups,advantage can be taken of PLA₂'s preference for anionic vesicles overzwitterionic vesicles. In selecting the backbone segments that bearthese functional groups, further advantage can be taken of fact that thenatural substrate of PLA₂ contains a phosphate group. Therefore,phosphodiester or phosphorothioate and other phosphate linked oligomericcompounds may be selected, providing a negatively charged compound forbinding with the positively charged interfacial binding site of PLA₂.

Compounds of the invention also include aromatic functional groups tofacilitate binding to the cleft of the PLA₂ enzyme. (see, Oinuma, etal., J. Med. Chem. 1991, 34, 2260; Marki, et al., Agents Actions 1993,38, 202; and Tanaka, et al., J. Antibiotics 1992, 45, 1071). Benzyl and4-hexylbenzyl groups are preferred aromatic groups. The compounds of theinvention can further include hydrophobic functional groups such astetraethylene glycol groups. Since the PLA₂ enzyme has a hydrophobicchannel, hydrophobicity is believed to be an important property ofinhibitors of the enzyme.

In certain embodiments of the invention, aminodiol monomer subunits areincorporated into libraries of oligomeric compounds and increasinglyless complex subsets of oligomeric compounds are identified incombinatorial screening techniques such as the above described SURF™technique by successive rounds of screens. The PLA₂ assay can beeffected using a combinatorial screening strategy such as the SURF™strategy. For this assay, the libraries of oligomeric compounds arescreened for inhibition of human type II PLA₂ enzymatic activity.Typically, these libraries contain about 100 to 100,000 differentcompounds.

Successive iterations of the SURF™ technique is effected to selectunique oligomeric compounds from the library. The libraries additionallycan be screened in other in vitro assays to determine further mechanismsof inhibition.

Upon identification of oligomeric compounds in a first phase ofscreening, further modifications can be made to the contents of thelibraries of oligomeric compounds. For example, if a first iteration ofscreening results in an active compound that contains a benzyl group,then in subsequent iterations of the screen this aromatic residue canthen be varied using substituted benzyl groups. In this way, structuralactivity is identified in a stepwise manner to define potent inhibitorsof the enzymatic activity.

To maximize the identification of a tight binding oligomeric inhibitorof PLA₂ via a combinatorial approach, an array of functional groupstypically are included in a randomized library.

Aminodiol monomer subunits can be linked with one another to formhomopolymeric structures or they can be linked with nucleotides and/orother moieties to form chimeric structures. For example, chimericstructures can be formed that include one or more regions or “stretches”of the monomer subunits of the invention joined to one or more regionsor “stretches” of naturally occurring or synthetic oligonucleotides orto other synthetic or natural oligomeric compounds such as peptides,peptoids, peptide nucleic acids, oligo and/or polysaccharides. Further,oligomeric compounds of the invention can be incorporated into chimericstructures along with the compounds disclosed in the patent applicationentitled “Monomeric Diols And Phosphate Linked Oligomeric compoundsFormed Therefrom,” Ser. No. 08/179,970 filed Jan. 11, 1994; which is theparent case for PCT/US95/0049, filed Jan. 11, 1995; and the patentapplication entitled “Oligonucleotide Mimics Having Nitrogen-ContainingLinkages,” Ser. No. 08/180,124, filed Jan. 11, 1994; which is the parentcase for PCT Application bearing attorney docket ISIS-1852, filed Jan.11, 1995. The foregoing patent applications are commonly assigned, andare incorporated herein by reference.

In one aspect of the invention, a combinatorial library of oligomericcompounds is synthesized by first attaching a plurality of aminodiolmonomer subunits having structure I, II, III, IV, V, VI, or VII, below,to a solid support.

The first aminodiol monomer subunits have base labile protecting groupsfor R₁ and a hydrogen or an alkyl group for R₂, if present. One of R₃and R₄ is an acid labile protecting group and the other is hydrogen e.g.a free hydroxyl. The free hydroxyl is reacted with a group on the solidsupport to effect the attachment of the aminodiol monomer subunit to thesolid support. Alternatively other bifunctional groups such as ethyleneglycol are attached to the solid support with many such compounds knownin the art and many commercially available. Solid supports are alsoavailable with linking groups previously attached, ready for use withouta derivatization step.

The base labile protecting group on the reactive amino group is removedby contacting with a base such as 10% piperidine in DMF or DBU(diazabicycloundecene) in pyridine, and a substituted carboxylic acid oranother electrophilic reagent (acid chloride, sulfonyl chloride, etc.)is then covalently linked to the backbone segment amino combinatorialsite using standard coupling methods (for examples see Bodansky, M.,Principles of Peptide Synthesis, 1984, Springer-Verlag, Berlin).Carbamates can be obtained by the treatment of the amine with anappropriate alkyl or aryl chloroformate. Carbamates can be obtained bythe treatment of the amine with an appropriate alkyl or arylchloroformate, in the presence of a catalyst such as pyridine. A urea orthiourea can be formed by reacting the backbone segment aminocombinatorial site with an isocyanate or isothiocyanate, or by treatmentwith carbonyl diimidazole followed by a second amine, in the presence ofbase. Sulfonamides can be prepared from the amine by the reaction with asulfonyl chloride in the presence of a base. The nitrogen can bealkylated by treatment with a halide such as the illustrative halides(benzyl bromide, 3-methylbenzyl bromide, 3-methoxybenzyl bromide or3-nitrobenzyl bromide) used in the examples below. A wide spectrum ofhalides can be used for this purpose. Additionally, amino compounds canbe functionalized by reaction with an aldehyde or ketone forming aSchiff base. The Schiff base is then reduced in the presence of areducing agent such as NaCNBH₃.

Functional groups that require protection are derivatized using acidlabile protecting groups which are stable to TCA. The acid labilehydroxyl protecting group is removed by treating with an acid (3% TCA indichloromethane) and the resulting free hydroxyl is reacted with asecond aminodiol monomer subunit H-phosphonate having a base labileprotecting group for R₁ and a hydrogen or an alkyl group for R₂, ifpresent. One of R₃ and R₄ is an acid labile protecting group and theother is an activated phosphite. The free hydroxyl of the first monomersubunit on the solid support is reacted with the activated phosphite ofthe second monomer subunit to form a phosphite linkage therebyincreasing the length of the growing oligomeric compound by one monomersubunit.

The phosphite linkage is oxidized to a phosphoramidate using a 10%solution of a primary or secondary amine in CCl₄/pyridine. The baselabile protecting group on the second monomer subunit is then removedwith piperidine as above. The resulting amino group is further reactedwith an activated functional group. If desired, additional monomersubunits can be added by repeating the synthetic steps described above.

Monomer subunits of the invention can be used to prepare oligomericcompounds having either preselected sequences or sequences determinedvia combinatorial strategies. One useful combinatorial strategy is theabove-noted SURF™ strategy, which is disclosed and claimed in U.S.patent application Ser. No. 749,000, filed Aug. 23, 1991, and PCTApplication US92/07121, filed Aug. 21, 1992, both of which are commonlyassigned with this application. The entire disclosure of theseapplications are herein incorporated by reference.

The following is an example of the methods used for the synthesis of asimple combinatorial library. The variable parameters which can becontrolled are the following: the backbone segments, the functionalgroups attached to the backbone segment amino combinatorial site, theamines attached at the phosphoramidate linkage, and the length of theoligomer. In the example described here a very simple library composedof only 32 molecules will be described. This library is prepared byusing two different backbone segments, A and B, two carboxylic acids Xand Y, and two amines, P and Q. 1) One begins by weighing equal amountsof solid supports derivatized with A and B. The two solid supports aremixed together by suspending the gels in DMF/dichloromethane (1:1) orother solvent, and mixing gently. If more than two backbone segments areused in the library, all the different supports are mixed together atthis stage. The mixture of supports is then treated with piperidine inDMF to remove the N-protecting group from both supports. 2) The mixtureis separated into a number of equal portions corresponding to the numberof reagents to be added in the next step, in this case two. To the firstmixture is added activated carboxylic acid X, to the is added activatedY. Once the reaction is complete, the solid supports are mixed as aboveto give an equimolar mixture of all four possible combinations. Themixture is then treated with trichloroacetic acid to remove the DMTprotecting groups on the monomer subunits and provide a free OH group.3) The mixture is separated into a number of equal portionscorresponding to the number of monomer subunits to be added, in thiscase two. To each mixture a monomer subunit H-phosphonate is thencoupled, the solid supports are again mixed, and the support is againdivided into equal portions corresponding to the number of amines to beadded, in this case two. 4) At this stage all eight possiblecombinations are present. Each portion is treated separately with asolution an amine, P or Q in CCl₄/Pyridine. The portions are thenpooled, treated with piperidine in DMF and split. 5) Each mixture of 16compounds on solid support treated with an activated carboxylic acid, Xand Y. The result is two unique pools of 16 compounds each, which canthen be deprotected to remove the terminal DMT group and any protectinggroups on the functionalities, and cleaved from the solid support. It isalso possible to add further combinatorial sites by coupling additionalmonomer subunits and extending the library molecules. We have used thefollowing nomenclature to describe mixtures of compounds in a concisemanner: X represents a fixed position, that is the backbone segment orfunctional group on the amino combinatorial site at that position isunique and serves to define the pool composition. N represents anequimolar mixture of all possible structures at a particular position.The subscript defines the synthetic step in the library synthesis. Inthe above example the library made was X₅N₄N₃N₂N₁, where X₅=X or Y, N₄is an equal mixture of P and Q, N₃ =A and B, N₂ X and Y, and N₁=A and B.In this cas the fixed position is the last one, but similar methods canbe used to fix any position in the library independently. It is alsopossible to have a single structure or functionality at any position.Once the most active moiety at position 5 is determined for a particularassay e.g. A, then two additional pools are synthesized having thisresidue in all cases. A second position is fixed and the remainderrandomized: for example A₅X₄N₃N₂N₁, where N¹⁻³ are as above and X₄ iseither P or Q. A unique structure is determined after 5 rounds ofsynthesis and screening. The advantage is uncovered when more than twopossible components are used in each position: If ten differentcomponents are available at each position then 100,000 unique structuresexist, yet only 5 rounds of synthesis and screening are necessary.

Illustrative of the SURF™ strategy is a 2′-O-methyl oligonucleotidelibrary (see, Ecker et. al., ibid.) shown in Table I, below. Table Idescribes the selection of a 2′-O-methyl oligonucleotide for binding toan RNA hairpin. The K_(D)'s, i.e., the binding constants, weredetermined by gel shift. “X” is used to indicate the position beingvaried and underlining is used to indicate positions that become fixedduring successive iterations of the SURF™ strategy.

TABLE I K_(D) (mM) Subsets X = A X = C X = G X = T Round 1 NNNNXNNNN  22 10 >100 >100 Round 2 NNNNCNXNN >10  4   >10   >10 Round 3NNXNCNCNN >10  0.5   >10   >10 Round 4 NNCXCNCNN >10  0.15   >10   >10Round 5 NNCCCXCNN    0.08 >1    0.4  >1 Round 6 NNCCCACXN    0.05 >0.5   0.08  >0.5 Round 7 NXCCCACAN  >0.1 >0.1    0.03  >0.1 Round 8NGCCCACAX    0.05  0.02    0.05    0.04 Round 9 XGCCCACAC    0.03  0.05   0.02    0.01

One aspect of the present invention is the inclusion of monomer subunitsof the invention in the above-described SURF™ strategy. The SURF™strategy is equally applicable to libraries of chemical compounds of thepresent invention in a completely parallel manner. Many other assays arealso used as the selection criteria to deduce a winning sequence withthe highest activity. The functional groups appended to the reactiveamino groups of the oligomeric compounds of the invention can be ofvarious structures that impart particular interactive properties to theoligomeric compounds. These chemical functional groups can effectinteractions of at least the following types: hydrogen-bond donors andacceptors, ionic, polar, hydrophobic, aromatic, electron donors andacceptors, pi bond stacking or metal binding. As a result of suchinteractions, the oligomeric compounds of the invention have uniqueproperties effecting the overall global shape, the conformational space,electron density, dipole moment and ability of the compound to interactwith enzyme pockets and other binding sites and other similarproperties.

To detect an active sequence generated via a combinatorial technique,the concentration of the active molecule is selected to be ofsufficiently great that the molecule can be detected within thesensitivity of the chosen assay. As will be recognized, the number ofunique structures within a subset produced via a combinatorial techniquedepends on the length of the oligomer and the number of differentfunctionalities employed. The number of structures is given by theproduct of the number components at each variable position. This isillustrated in Table II. Table II also indicates the concentration ofeach sequence when the subset concentration is 100 uM, a typicalhigh-test concentration. We have found that the complexity of thelibrary can be based upon an estimate of the expected IC₅₀ (i.e., aconcentration at which 50% of enzyme activity is inhibited) that isdesirable in a final oligomeric compound. For an expected IC₅₀ of 100nM, the complexities shown in Table II are acceptable, that is, thelibraries shown in Table II have complexities that would allow detectionof a unique sequence with an IC₅₀ of about 100 nM or less.

TABLE II Complexity of Libraries Variable Positions Sequences nM EachSequence (Mer) Per Subset At 100 μM Subset 5 Components 4-mer 125 8005-mer 625 160 6 Components 4-mer 216 463 5-mer 1,296    77 7 Components4-mer 343 291 8 Components 4-mer 512 195 10 Components 4-mer 1,000   100

The functional groups or components can also be referred to as“letters.” The use of such terminology reflects the fact that thedifferent functional groups on the compounds of the invention arepositioned in sequences (either predetermined or by random selection)much like letters of the alphabet, hence the term “letter.” Theseletters can be “reactive” or “non-reactive.” By “reactive,” it is meantthat they will interact with a target molecule in some manner (that neednot but can be predefined). By non-reactive,” it is meant that they arenot designed to primarily interact with a target molecule, and in factwhile they may interact with the target molecule, the primary purpose ofthe non-reactive moieties are to impart other properties to the moleculesuch as, but not necessarily limited to, effecting up-take,distribution, metabolism or identification.

A further advantage of this invention is the ability to synthesizeoligomeric compounds that, in addition to or in place of variability inthe sequences of the diverse functional groups, have an asymmetricsequence of monomer subunits. Stated otherwise, the monomer subunits canalso vary within the oligomeric compounds. This is easily accomplishedby using different aminodiol monomer subunits that eventually become thebackbone of the oligomeric compounds.

The oligomeric compounds of the invention can be used in diagnostics,therapeutics and as research reagents and kits. They can be used inpharmaceutical compositions by including a suitable pharmaceuticallyacceptable diluent or carrier. In preferred embodiments, the compoundsof the invention act as inhibitors of enzymes such as phospholipase A₂;as inhibitors of pathogens such as virus, mycobacterium, bacteria (gramnegative and gram positive), protozoa and parasites; as inhibitors ofligand-receptor interactions such as PDGF (platelet derived growthfactor), LTB₄ (leukotriene B₄), IL-6 and complement C5_(A); asinhibitors of protein/protein interactions including transcriptionfactors such as p50 (NF_B protein) and fos/jun; for the inhibition ofcell-based interactions including ICAM induction (using inducers such asIL1-β, TNF and LPS) and as herbicides and insecticides. In otherpreferred embodiments, the compounds of the invention are used asdiagnostic reagents for each of the above noted biological entities, andas reagents in assays and as probes. In other preferred embodiments, thecompounds of the invention are used to chelate heavy metals and asimaging agents.

The functional groups can be selected based on chain length, i.e. shortversus long, based on the use of a ring versus a linear group, use of anaromatic versus aliphatic group, use of a functionalized group versus anon-functionalized group, to mention only a few of the wide variety ofchemical functional groups available. Indeed simply varying functionalmoieties, e.g. acid, alcohol, aldehyde, amide, amine, amidine, azo,azoxy, double bond, ether, ethylene oxide, guanidine, halide, haloalkyl,hydrazine, hydroxylamine, ketone, mercaptan, nitrate, nitrile, nitro,nitroso, quaternary nitrogen, sulfide, sulfone, sulfoxide, triple bond,urea, etc. on a single backbone segment amino combinatorial site, e.g. as:Lmple alkyl group, yields a vast array of diversity functions. Whenthis is expanded to include placement of such varied functional moietieson a broad platform of backbones, e.g. a series of alkyl compounds, aseries of aryl compounds, a series of alicyclic compounds, etc., thepotential for a vast array of chemical functional groups is apparent.Other chemical functional groups include alkyl, alkenyl, alkynyl,alicyclic and substituted alkyl, alkenyl, alkynyl, alicyclic; aryl andsubstituted aryl; aralkyl, substituted aralkyl, heterocycles,nucleobases such as pyrimidines and purines, metal chelating groups andmoieties as found in the α-position of amino acids, such as those shownbelow:

CH₃—

HO—CH₂—

C₆H₅—CH₂—

HO—C₆H₄—CH₂—

CH₃—CH₂—S—CH₂—CH₂—

HO—CH₂—CH₂—

CH₃—CH₂(OH)—

HO₂C—CH₂—NHC(═O)—CH₂—

HCO₂—CH₂—CH₂—

NH₂C(═O)—CH₂—CH₂—

(CH₃)₂—CH—

(CH₃)₂—CH—CH₂—

CH₃—CH₂—CH₂—

H₂N—CH₂—CH₂—CH₂—

H₂N—C(NH)—NH—CH₂—CH₂—CH₂—

H₂N—C (═O) —NH—CH₂—CH₂—CH₂—

CH₃—CH₂—CH (CH₃)—

CH₃—CH₂CH₂—CH₂-CH₂—

H₂N—CH₂—CH₂—CH₂—

HS—CH₂—

HO₂C—CH (NH₂)—CH₂—S—S—CH₂—

CH₃—CH₂—

CH₃—S—CH₂-CH₂—

The various combination of reactions that can be effected utilizing theteaching of this invention can be further demonstrate by use of alogical flow scheme. This flow scheme is set forth in FIGS. 1A and 1B.

Referring to the figures, at the start 10 of the synthesis, an aminodiolmonomer subunit or group of aminodiol monomer subunits is/are selected.The selected monomer or monomers are processed at process step 12wherein the aminodiol monomer subunit or subunits is/are attached to asolid support as per Examples 26, 30, 35, 45, 46, 57, 55, and 61, setforth below. The aminodiol monomer subunit may be attached to the solidsupport via a H-phosphonate diester linkage, through a succinyl linkage,or through an ethylene-glycol linkage. Many other types of linkages areknown in the art and are amenable to the present invention. A group ofaminodiol monomer subunits may be advantageously attache at this step byutilizing a split bead synthesis. The process steps effected at processstep 12 can be otherwise characterized as a selecting and attachingprocedure depicted by box 102.

From the procedure box 102, the path of the flow scheme intersectsconvergent point 14. Convergent point 14 represents the point in theiterative process of the invention wherein growing oligomeric compoundsfrom later steps are reintroduced via this iterative process to undergofurther oxidation, amine group functionalization and elongation asdiscussed below.

Downstream of the convergent point 14, the processes of the inventionfurther include an oxidization step effected via procedures included inthe general oxidization procedure depicted by box 104. At decision block16, within the oxidization procedure generally depicted at box 104, theproduct of process step 12 (or the growing oligomeric compoundsintroduced at convergent point 14) is (are) either converted byoxidation from p^(III) to p^(v) compounds via the positive branch ofthis step or the p^(III) phosphite linkages are not oxidized and thecompounds are maintained with p^(III) linkages and are taken to the nextdecision step via the negative branch of decision block 16 leading toconvergent point 42. For those products of process step 12 (or thegrowing oligomeric compounds introduced at convergent point 14) whereinoxidization is desired during this iteration of the process (thosemoving along the positive branch of decision block 16), at decisionblock 18 they are either converted to phosphoramidates via the positivebranch leading from decision block 18 or they are converted intophosphorothioates or phosphodiesters via the negative branch leadingfrom decision block 18.

At decision block 20, growing oligomeric compounds are either oxidizedto phosphodiesters at process step 24 via the positive branch ofdecision block 20 or they are oxidized to phosphorothioates at processstep 25 via the negative branch of decision block 20. Oxidation toeither the phosphorothioate or phosphodiester, as illustrated in Example69, can be effected simultaneously on multiple positions on the samegrowing oligomeric compound, as for instance if the negative branch wastaken at decision block 16 for one or more previous iterations of theprocess. If there are previous unoxidized sites and if the positivebranch of decision block 16 is selected during the current iteration,multiple monomeric units are oxidized simultaneously. The resultingphosphodiesters and phosphorothioates converge at convergent point 38and from there lead to convergent point 40.

Following the positive branch from decision block 18, growing oligomericcompounds at decision block 22 can be converted to phosphoramidates byone of three process steps. First, the decision to effect substitutionusing a single amine is effected at decision block 22. Growingoligomeric compounds following the positive branch from decision block22 are converted at process step 26 using a single amine in solution asillustrated in Example 67, to give phosphoramidate linkages havinguniform substituents. Growing oligomeric compounds following thenegative branch from decision block 22 are combinatorialzed by one oftwo processes. Growing oligomeric compounds following the negativebranch from the competitive combinatorialzation decision block 28 arecombinatorialzed at process step 30 by a split bead synthesis. Growingoligomeric compounds following the positive branch of decision block 28are combinatorialized at process step 32 using a mixture of amines asillustrated in Example 67 Method B.

Growing oligomeric compounds that may have been oxidized (via thepositive branch of decision block 16) or may not have been oxidized (viathe negative branch of decision block 16) converge at convergent point42 within the general oxidization procedure box 106. From convergentpoint 42 the process is continued on FIG. 1B via connector point 44 thatconnects FIG. 1A and FIG. 1B. A further connector point, point 46, leadsback from FIG. 1B to FIG. 1A.

At decision block 48 within the generalized functionalization proceduredepicted by box 106, following the positive branch of decision block 48,the growing oligomeric compounds, at process step 50, are deblocked toexpose a free amino site on the growing oligomeric compound. This freeamino site is now available for functionalization. Alternatively,following the negative branch from decision block 48, amino deblockingand functionalization is bypassed. Following the negative branch ofdecision block 48, the amine protecting groups are maintained and thegrowing oligomeric compound is taken directly to convergent point 66.

If in one or more earlier iterations of the process, the negative branchof decision block 48 was selected, there will be multiple amino sitesthat now are protected and can be deblocked for functionalization. Thusfunctionalization can be effected at one or at more than one site duringany one iteration of the functionalization procedure generally depictedby box 106.

Following the positive branch of the functionalization proceduredecision block 48, after deblocking at process step 50, severalalternatives are available for functionalization of the resulting freeamino site(s). At decision block 52, functionalization with a singlefunctional group or with multiple functional groups is queried.Following the positive branch from decision block 52, functionalizationis effected with a single functional group. This functionalization canbe performed on a single position or, as noted above, if thefunctionalization procedure was bypassed in prior iterations of theprocess, multiple amino positions in a growing oligomeric compound orcombinatorial library of such compounds can be functionalizedsimultaneously. The negative branch of decision block 52 leads todecision block 56 where the deprotected amino site on the growingoligomeric compound can be functionalized with multiple reagents via twodifferent combinatorialization procedures. Following the negative branchfrom decision block 56, combinatorialization of the free amino site(s)of the growing oligomeric compound(s) is effected at process step 58using a split bead synthesis. Following the positive branch fromdecision block 56, combinatorialization of the free amino site(s) iseffected at process step 60 by using a mixture of compounds e.g.carboxylic acids or acid halide as, for example, via Examples 63 and 65.

The products of process steps 58 and 60 converge at convergent point 62and these in turn converge with the products of process step 54 atconvergent point 64. Both of these points converge at convergent point66 where intermediates that were shunted by the deblocking process step50 via the negative branch of the decision block 48 also converge.

A query is made at decision block 68 to discontinue synthesis or tofurther elongate the growing oligomeric compound. If the currentoligomeric compounds is of sufficient length, at decision block 68, adecision to effect final deblocking, generally depicted by box 108, ismade. Following the negative branch of decision block 68, finaldeblocking is effected at process step 72 to give the final product orproducts 78.

If further extension of the growing oligomeric compound is desired, thepositive branch of decision block 68 is followed. This leads to theextension procedure generally depicted by box 110. To effect oligomericcompound elongation, at process step 70 a hydroxyl protecting group isremoved and at process step 74 a further aminodiol monomer subunit isadded. The growing oligomeric compound is now re-introduced atconversion point 14 (via connecting point 46) into the process loop fora further iteration of synthesis depicted in FIG. 1A. Here the processof selection of paths independently leading to or bypassing oxidationand/or functionalization is then repeated.

The process of the invention can generally be characterized as firstselecting and attaching one or more monomer subunits to a support. Thiscorresponds generally to procedure box 102 of the figures. For themoment bypassing the oxidization procedure, generally depicted by box104, and the functionalization procedure, generally depicted by box 106,the hydroxyl blocking group of the monomeric subunits can be removed anda further iteration of the addition of monomer units effected. This isaccomplished by the extension procedure generally depicted by box 110.

If during this iteration of the process, oxidization of the phosphatelinker is desired, oxidization will be effected via the oxidizationprocedure generally depicted by box 104. Further if prior to the nextiteration but post the oxidization procedure, functionalization of theamino group of the last monomer subunit(s) added is desired,functionalization is effected via the functionalization proceduregenerally depicted by box 106.

If the growing oligomeric compound or compounds is/are of sufficientlength, synthesis is halted and the compound or compounds are deblockedand cleaved from their supports via the final deblocking procedure,generally depicted by box 108.

It is of course recognized that the oxidization step of the firstmonomer subunit can be effected or oxidization of each subsequentlyadded monomer subunit can be effected during the iteration of theprocess when they are added. Alternatively, oxidization need not beeffected for each monomeric subunit added but can be effected duringsome subsequent iteration of the process. It is further recognized thatfunctionalization of the amino site can be effected for the firstmonomer subunit and it can be effected for each added monomer subunit.Alternatively, functionalization need not be effected for each monomericsubunit added but can be effected during some subsequent iteration ofthe process.

EXAMPLE 1 N-Fmoc-trans-4-Hydroxy-L-Proline

trans-4-Hydroxy-L-proline (5.00 g, 38.2 mmol) and NaHCO₃ (8.00 g, 95.2mmol) were suspended in 150 ml H₂O/Dioxane (1:1). Fluorenylmethylchloroformate (11.4 g, 44.0 mmol) in 25 ml toluene was added dropwise.The temperature of the reaction was not allowed to rise above 25° C.during the addition. The mixture was stirred vigorously overnight, andthen quenched with 50 ml saturated NaHCO₃ solution and 50 ml water. Thesolution was then extracted with 100 ml diethyl ether. The aqueous layerwas acidified to pH 1 with concentrated HCl, and extracted twice withethyl acetate, and the organic extracts washed with brine. The solutionwas dried with MgSO₄, filtered and the solvent removed in vacuo. Thepure product crystallized from the concentrated solution. Yield: 13.4 g(100%). ¹H NMR: (CDCl₃, 200 MHz) δ 7.75-7.15 (8H, m, ArH), 4.55-4.05(5H, m, ArCHCH₂, H2, H4), 3.65-3.45 (2H, m, 2 H5), 2.35-2.10 (2H, m, 2H3).

EXAMPLE 2 N-Fmoc-3-Hydroxypyrrolidine-5-Methanol

To a solution of N-Fmoc-trans-4-hydroxy-L-proline (13.4 g, 38.1 mmol) in250 ml THF was added borane-methyl sulfide (78 mmol, 5.78 g, 7.22 ml)dropwise at room temperature. After the evolution of H₂ had ceased, thesolution was heated to reflux with mechanical stirring. After 1 hour awhite precipitate had formed. Methanol was carefully added, and theresulting solution refluxed for a further 15 minutes. The solution wascooled to room temperature, the solvents evaporated under reducedpressure, and the residual gum coevaporated with 2×100 ml methanol. Theresulting crystalline product weighed 12.0 g (35.3 mmol, 93%). ¹H NMR:(CDC13, 200 MHz) δ 7.85-7.25 (8H, m, ArH), 4.50-4.10 (5H, m, ArCHCH2,H3, H5), 3.80-3.40 (4H, m, 2 H2, 2 H6), 2.15-1.95 (1H, m, H2a),1.80-1.60 (1H, m, H2b).

EXAMPLE 3 N-Fmoc-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

The diol, N-Fmoc-3-hydroxypyrrolidine-5-methanol (10.59 g, 31.2 mmol)was coevaporated with dry pyridine (2×50 ml), redissolved in 200 ml drypyridine, and cooled in a ice bath. Dimethoxytrityl chloride (11.0 g,32.5 mmol) was added in portions over 30 min, and the solution stirredat 0° C. overnight. Methanol was then added (10 ml), and the solventremoved under reduced pressure. The resulting gum was redissolved intoluene (100 ml), filtered to remove the pyridinium hydrochloride andtaken to dryness again. The residue was dissolved in CH₂Cl₂ (300 ml),washed with 150 ml 0.1 M citric acid solution, 150 ml sat NaHCO₃, brine,and dried with MgSO₄ followed by evaporation. The residue wascrystallized from methanol and dried to give (15.4 g, 23.9 mmol, 77%).

EXAMPLE 4 5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

To a solution N-Fmoc-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine(3.40 g, 5.30 mmol) in 15 ml DMF was added piperidine (1.09 ml, 0.935 g,11.0 mmol). The solution was stirred at room temperature for 1 hour,water (100 ml) added, and the aqueous solution extracted with ethylacetate (2×75 ml). The organic extracts were washed with aqueous NaHCO₃,brine, dried with MgSO₄ and evaporated. The residue was purified byflash column chromatography using a gradient of 1→3% MeOH in CH₂Cl₂containing 0.5% triethylamine. Pure product was obtained (1.86 g, 84%).¹H NMR: (CDCl₃, 200 MHz) δ 7.42-6.80 (13 H, ArH), 4,35 (1H, m, H5), 3.77(6H, s, 2 OCH₃), 3.62 (1H, m, H3), 3.13-2.88 (4H, m, 2 H6, 2 H2), 1.87(1H, q, H4a), 1.65 (1H, m, H4b).

EXAMPLE 5N-(Phenylacetyl)-5-Dimethoxytrityloxymethyl-3-Hydroxypyrrolidine

Phenylacetic acid (1.50 g, 11 mmol) and HOBT (1.63 g, 12 mmol) weredissolved in 100 ml CH₂Cl₂ and EDC (15 mmol, 2.88 g) was added. After 15min, 5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine was added, followedby DIEA (20 mmol, 3.5 ml). The reaction was stirred until the startingmaterial was consumed, and quenched with 10 ml NaHCO₃. The mixture wasextracted twice with ethyl acetate, washed with NaHCO₃, brine, driedwith MgSO₄, and evaporated. The product was purified by flashchromatography to give 4.0 g product (75%). ¹H NMR: (CDCl₃, 200 MHz) (2rotamers, 3′-O-TMS) d 7.43-7.13, 6.88-6.74 (13 Ar-H), 4.67, 4.49, 4.37,4.13 (4 m, 2H, H3, H5), 3.78 (s, 6H, OCH₃), 3.78-3.50 (m, 2H, H2a, b),3.66, (s, 2H, CH₂Ar) 3.35 (q, 1H, H6a), 3.12 (m, 1H, H6b), 2.14-1.70 (m,2H, H4a, b), 0.10 (d, 9H, OSi(CH₃)₃).

EXAMPLE 6 Succinic Acid Fluorenylmethyl Ester

Fluorenemethanol (10.0 g, 51.0 mmol) was dissolved in 150 ml CH₂Cl₂, andsuccinic anhydride (5.6 g, 56 mmol) was added. The solution was stirredfor 6 h, and a further portion of succinic anhydride (2.5 g, 25 mmol)was added, and stirring continued overnight. The reaction appearedcomplete by TLC. The solvent was then removed, and the residue extractedwith ethyl acetate, washed with 1% HCl, water, brine, dried (MgSO₄) andevaporated to an oil which crystallized on standing. A quantitativeyield of crude product was obtained which was used without furtherpurification.

EXAMPLE 7 (N1-Thymine)-2-Acetic Acid

Methyl bromoacetate (25.5 g, 15.2 ml, 160 mmol) was added to asuspension of K₂CO₃ (44.2 g, 320 mmol) and thymine (20.2 g, 160 mmol) in500 ml dry DMF with stirring overnight. The suspension was filtered andthe solvent removed under reduced pressure. The residue was suspended in120 ml H₂O and 30 ml 4 N HCl, stirred for 30 minutes and filtered again.The solid was suspended in 250 ml H₂O, to which was added 100 ml 2.5 MNaOH. The solution was heated to boiling, cooled and acidified to pH 1with concentrated HCl. The precipitate was dried in vacuo to give 1-3.6g (73.6 mmol, 46%) pure product. ¹H NMR: (DMSO-d6, 200 MHz) δ 7.48 (s,1H, H6), 4.37 (s, 2H, CH₂), 1.76 (s, 3H, CH₃)

EXAMPLE 8 N-Fmoc-3-Aminopropionic Acid

Sodium bicarbonate (2.52 g, 30 mmol) and 3-aminopropionic acid (1.00 g,11.2 mmol) were dissolved in 50 ml water and 50 ml dioxane was added. Asolution of fluorenylmethyl chloroformate (3.10 g, 12.0 mmol) in 50 mldioxane was added dropwise with stirring. After 6 hours the solution wasdiluted with water (100 ml) and saturated bicarbonate solution (50 ml),extracted once with diethyl ether, and the aqueous layer acidified to pH2 with concentrated HCl. The cloudy solution was extracted with ethylacetate (2×100 ml), washed with brine and dried with MgSO₄. Afterevaporation a mixture of the title product and the peptide dimer wasobtained. The pure product was obtained by flash chromatography. ¹H NMR:(CDCl₃, 200 MHz) δ 7.95-7.26 (8H, m, ArH), 7.40-7.15 (3H, m, CHCH₂O),3.20 (2H, t, J=8 Hz, CH₂N), 2.40 (2H, t, J=8 Hz, HOOCCH₂).

EXAMPLE 9 N-Imidazolyl-2-Acetic acid

Imidazole (3.7 g, 54 mmol) was added to a suspension of sodium hydride(2.6 g of a 60% dispersion in oil, 60 mmol) in 50 ml dry THF.Bromoacetic acid (3.4 g, 24 mmol) was then added and the mixture stirredovernight. Water (1 ml) was then added and the solvent removed underreduced pressure. The residue was taken up in water (50 ml, pH >10),extracted with ether and the organic layer discarded. The aqueous layerwas acidified to pH 1 with concentrated HCl and extracted again withether. The aqueous layer was evaporated to dryness. The oily residue wasdissolved in absolute ethanol (EtOH) to precipitate NaCl, andrecrystallized from acetone/methanol to give 1.22 g (7.5 mmol, 30%) pureproduct as the hydrochloride. ¹H NMR: (DMSO-d6, 200 MHz) δ 9.20 (s, H2),7.76 (d, J=1.5 Hz), 7.69 (d, J=1.5 Hz), 5.20 (s, CH₂).

EXAMPLE 10 (9-Adenine)-2-Acetic Acid Ethyl Ester

Sodium hydride (8.20 g 60% in oil, 205 mmol) was added to a suspensionof adenine (25.0 g, 185 mmol) in 500 ml DMF. After vigorous stirring for2 hours using a mechanical stirrer, H₂ evolution stopped and a thickslurry was obtained. Ethyl bromoacetate (55.6 g, 36.9 ml, 333 mmol) wasadded dropwise over 3 hours, and stirring continued for a further 1hour. Water (10 ml) and H₂SO₄ were added to pH 4. The solvent wasevaporated and the residue suspended in 500 ml H₂O, filtered and washedwith water. The residue was recrystallized from 400 ml ethanol to give23.8 g (108 mmol, 58%) pure product.

EXAMPLE 11 (N6-Benzoyl-9-Adenine)-2-Acetic Acid

To a suspension of (9-adenylyl)-2-acetic acid ethyl ester (6.06 g, 27.4mmol) in 250 ml dry pyridine was added benzoyl chloride (9.60 ml, 11.6g, 82 mmol), and the solution stirred for 4 hours at room temperature.Methanol (25 ml) was added and the solvents evaporated. The residue wasdissolved in ethyl acetate (2×250 ml), washed with 0.1 N HCl, H₂O,saturated NaHCO₃, brine, and dried with Na₂SO₄. The organic extractswere evaporated and the solid residue was redissolved in 250 ml THF at0° C., to which was added 100 ml 1M NaOH. The solution was stirred at 0°C. for 1 hour and acidified to pH 1 with concentrated HCl, and theaqueous portion extracted once with ether. The product, which began tocrystallize almost immediately, was collected by filtration to yield4.96 g (61%). ¹H NMR: (DMSO-d6, 200 MHz) δ 8.86, 8.84 (d, H2, H8), 8.1(d, 2H, J=7.0 Hz, ArH), 7.69-7.58 (m, 3H, Ar-H), 5.22 (s, 2H, CH₂).

EXAMPLE 12 N-4-Benzoylcytosine

Cytosine hemihydrate (12.0 g, 100 mmol) was coevaporated with pyridineand resuspended in 250 ml dry pyridines. Benzoyl chloride (58 ml, 70.3g, 500 mmol) was added dropwise (exothermic). The solution was stirredat RT overnight, and water (50 ml) carefully added. The solvent wasevaporated, and the residue dissolved in 700 ml H₂O containing 55 gNaOH. The solution was stirred for 1 h after complete dissolution of thematerial. Concentrated HCl was then added to pH 4.0, the whiteprecipitate was collected and boiled in 1 liter EtOH, cooled to RT andfiltered to give 16.1 g product (75%).

EXAMPLE 13 N-4-Benzoyl-1-Cytosine-2-Acetic Acid

To a suspension of N-4-Benzoylcytosine (15.0 g, 69.7 mmol) and K₂CO₃(9.7 g, 70 mmol) in 500 ml DMF was added methyl bromoacetate (6.6 ml,10.7 g, 70 mmol). The suspension was stirred vigorously for 3 days,filtered and evaporated. The residue was treated with water (120 ml),and 10 ml 4N HCl for 15 min, and the solid collected by filtration. Theresidue was resuspended in 120 ml water, and 60 ml 2N NaOH added. Thesuspension was stirred at RT for 45 min, until all the solids haddissolved. The solution was acidified to pH 2 with conc HCl, filtered,and the solid dried in vacuo at 60° C. to give 11.6 g product (61%).

EXAMPLE 14 N-2-Isobutyroyl-9-Guanine-2-Acetic Acid

To a suspension of 2-amino-6-chloropurine (10 mmol) and K₂CO₃ (15 mmol)in DMF (25 ml) is added ethyl bromoacetate (10 mmol). The mixture isstirred vigorously for 24 hours, filtered and the solvent evaporated.The residue is resuspended in 25 ml pyridine and isobutyroyl chlorideadded (20 mmol). After stirring for 18 hours, water is added and thesolvent removed. The residue is suspended in 1N HCl and heated to refluxfor 1 hour. The suspension is then cooled to 0° C., NaOH added to pH 12,and the suspension stirred for 1 hour. The solution is acidified to pH3, and the product is collected by filtration.

EXAMPLE 15 Benzyl 3,6,9,12-Tetraoxatridecanoate

Triethyleneglycol monomethyl ether (10 mmol) and benzyl bromoacetate (11mmol) are added to a suspension of anhydrous K₂CO₃ (15 mmol) in 50 mlanhydrous DMF. The suspension is stirred at room temperature overnight.Water is added and the emulsion is extracted with ethyl acetate (3×200ml), washed with water, brine, and dried with MgSO₄. The solvent isevaporated and the residual oil purified by flash chromatography to givethe title compound.

EXAMPLE 16 3,6,9,12-Tetraoxatridecanoic Acid

Benzyl-3,6,9,12-Tetraoxatridecanoate (5 mmol) is dissolved in methanol(50 ml) and 10% palladium on carbon is added (100 mg catalyst/mmol). Thesuspension is shaken under 30 psi H₂ until the starting material isconsumed. The suspension is filtered through a short pad of Celite,washed thoroughly with methanol, and the solvent evaporated. The productis used directly without purification.

EXAMPLE 17 Benzyl Bis-[(2-Pyridyl)-2-ethyl]-Aminoacetate

To a suspension of K₂CO₃ (15 mmol) in 25 ml DMF was added2,2′-bis(2-pyridylethyl)-amine (10 mmol) followed by benzyl bromoacetate(12 mmol). The suspension was stirred for 4 hours at room temperature.Water was then added, and the suspension extracted with ethyl acetate(2×100 ml), washed with 5% Na₂CO₃, water, brine, dried with MgSO₄ andthe solvents removed. The product was obtained as an oil in quantitativeyield. Product was identified by NMR.

EXAMPLE 18 Bis(2-(2-Pyridyl)ethyl)-Aminoacetic Acid

Benzyl bis-[(2-pyridyl)-2-ethyl]-aminoacetate (5 mmol) is dissolved inmethanol (50 ml) and 10% palladium on carbon is added (100 mgcatalyst/mmol). The suspension is shaken under 30 psi H₂ until thestarting material is consumed. The suspension is filtered through ashort pad of Celite, washed thoroughly with methanol, and the solventevaporated. The product is used directly without purification.

EXAMPLE 19 N-Carbazolyl-2-Acetic Acid

The title compound is prepared as per Example 13 using carbazole as thestarting heterocycle.

EXAMPLE 20 N-Pyrrolyl-2-Acetic Acid

The title compound is prepared as per Example 13 using pyrrole as thestarting heterocycle.

EXAMPLE 21 N-Trifluoroacetyl-Glycine Triethylammonium Salt

To a suspension of glycine (1.50 g, 20 mmol) in 100 ml dry methanol wereadded triethylamine (3.5 ml, 2.5 g, 25 mmol) and ethyl trifluoroacetate(3.0 ml, 3.55 g, 25 mmol). The mixture was stirred overnight to give ahomogeneous solution. The solvents were removed and the resulting oilcoevaporated with toluene to remove traces of methanol. The product wasused without purification.

EXAMPLE 22 2-O-(Dimethoxytrityl)ethanol

A solution of ethylene glycol (2.45 ml, 44 mmol) in dry pyridine (25 ml)was cooled to 0° C. in an ice bath. Excess triethylamine (7 ml) and4-dimethylaminopyridine catalyst (120 mg, 1 mmol) was added followed bythe slow addition of dimethoxytrityl chloride (7.42 g, 21.9 mmol) over30 minutes. The mixture was stirred at 0° C. for 1 hr and then roomtemperature for 1 hr. The resulting solution was quenched with methanoland evaporated to dryness under reduced pressure. The residue wasdissolved in saturated NaHCO₃ and extracted with EtOAc. The EtOAcextracts were washed with cold saturated sodium bicarbonate and brine.The organic phase is separated, dried over sodium sulfate, filtered andevaporated under reduced pressure. The resulting residue is purified byflash column chromatography on silica gel using ethyl acetate-hexanes(gradient 10 to 20%). The title compound was isolated to yield 5.53 g(70%). ¹H NMR: (CDCl₃) δ 7.50-7.20, 6.90-6.80 (m, 13 H, ArH), 3.80 (s, 6H, OCH₃), 3.75 (t, 2H, CH₂OH), 3.25 (t, 2H, DMTOCH₂).

EXAMPLE 23 2-Dimethoxytrityl Ethanol Hemisuccinate Triethylanmonium Salt

A solution of 2-O-(dimethoxytrityl)ethanol (1.0 g, 2.77 mmol),triethylamine (0.4 ml, 3 mmol), and 4-dimethylaminopyridine catalyst(120 mg, 1 mmol) in dry dichloroethane was treated with succinicanhydride (410 mg, 0.41 mmol). The mixture was stirred at 50° C. for 1.5hr and then cooled to room temperature. The mixture was kept at roomtemperature for 16 hrs. The mixture is filtered and the filtrate waspurified by silica gel flash column chromatography usingchloroform-methanol-triethylamine to yield the title compound as atriethylammonium salt. ¹H NMR: (CDCl₃) δ 7.50-7.20, 6.90-6.80 (m, 13 H,ArH), 4.26 (t, 2H, CH₂OCO), 3.80 (s, 6 H, OCH₃), 3.25 (t, 2H, DMTOCH₂),3.05 (q, 6H, N(CH₂CH₃)₃), 2.70 (m, 4H, OOCCH₂CH₂COO), 1.25 (t, 9H,N(CH₂CH₃)₃.

EXAMPLE 24 2-O-(Dimethoxytrityl)ethoxyphosphonic Acid

A solution of imidazole (4.29 g, 63 mmol)in dry acetonitrile at 0° C.(100 ml) was treated dropwise with PCl₃ (1.77 ml, 20.3 mmol) over aperiod of 30 minutes. The resulting solution is further treated withtriethylamine (9.06 ml, 65 mmol). To the thick slurry was added2-O-(dimethoxytrityl)ethanol (2.10 g, 5.81 mmol) in anhydrousacetonitrile (150 ml) slowly over a period of 30 minutes. The mixture isallowed to warm to room temperature and stirred for 15 minutes. Themixture is quenched with 1M TEAB and the mixture is evaporated in vacuoto a minimum volume and extracted with dichloromethane (2×150 ml). Thedichloromethane extracts are washed with TEAB and evaporated in vacuo.The residue was purified by flash column chromatography using a gradientof 0% to 5% methanol in dichloromethane/1% triethylamine to yield 1.3 gpurified material (43%). ¹H NMR: (CDCl₃) δ 7.50-7.20, 6.90-6.80 (m, 13H, ArH), 6.96 (d, 1H, J_(PH)=624 Hz, PH), 4.06 (m, 2H, CH₂OP), 3.80 (s,6 H, OCH₃), 3.25 (t, 2H, DMTOCH₂), 3.05 (q, 6H, N(CH₂CH₃)₃), 1.25 (t,9H, N(CH₂CH3)₃). ³¹P NMR (CDCl₃); 5.89.

EXAMPLE 25 Synthesis of 2-O-(Dimethoxytrityl)-ethylsuccinate Half Ester

A solution of 2-O-(dimethoxytrityl)ethanol (1.0 g, 2.77 mmol),triethylamine (0.4 ml, 3 mmol), and 4-dimethylaminopyridine catalyst(120 mg, 1 mmol) in dry dichloroethane was treated with succinicanhydride (410 mg, 0.41 mmol). The mixture was stirred at 50° C. for 1.5hr and then cooled to room temperature. The mixture was kept at roomtemperature for 16 hrs. The mixture is filtered and the filtrate waspurified by silica gel flash column chromatography usingchloroform-methanol-triethylamine to yield the title compound as atriethylammonium salt. ¹H NMR: (CDCl₃) δ 7.50-7.20, 6.90-6.80 (m, 13 H,ArH), 4.26 (t, 2H, CH₂OCO), 3.80 (s, 6 H, OCH₃), 3.25 (t, 2H, DMTOCH₂),3.05 (q, 6H, N(CH₂CH₃)₃), 2.70 (m, 4H, OOCCH₂CH₂COO), 1.25 (t, 9H, N(CH₂CH₃)₃.

EXAMPLE 26 Derivatization of LCAA CPG With2-O-(Dimethoxytrityl)-ethylsuccinate Half Ester

2-O-(Dimethoxytrityl)ethylsuccinate half ester triethylammonium salt(135 mg) was dissolved in dichloromethane (5 ml).4-Dimethylaminopyridine catalyst (40 mg, 0.2 mmol) was added followed bytoluene diisocyanate (0.029 ml, 0.2 mmol). The mixture was shaken for 18min. Long chain alkyl amine controlled pore glass (LCAA CPG) (1.0 g) wasadded and the mixture was shaken with the exclusion of light for 16 hrs.The mixture was filtered and washed with dichloromethane and thendiethylether (3×10 ml each). The CPG was shaken for 16 hrs inpyridine/water (4:1), filtered, and rinsed with pyridine (5×5 ml). A 10mg sample of the dried CPG was treated with 3% trichloroacetic acid indichloromethane. The presence of the trityl ion qualitatively verifiedthe derivatization. The loading was measured to be 30 μmol/g bymeasuring the absorbance of the dimethoxytrityl cation.

EXAMPLE 27 Synthesis of 10-O-(Dimethoxytrityl)-1-decanol

A solution of decane-1,10-diol in dry pyridine and containing excesstriethylamine is treated with one equivalent of dimethoxytrityl chloridefor a period of six hours. The resulting solution is evaporated todryness under reduced pressure, the residue redissolved in methylenechloride and the solution washed with cold saturated sodium bicarbonate,water and brine. The organic phase is separated, dried over sodiumsulfate, filtered and again evaporated under reduced pressure. Theresulting residue is flash-chromatographed on silica gel using ethylacetate-hexanes to isolate the purified product. Characterization byH-NMR yields signals for the DMT group (multiplet, 8.0-7.0 ppm), thedecane group (multiplets, 1.2-4.0 ppm) and the alcohol (variable).

EXAMPLE 28 Synthesis of 10-O-(Dimethoxytrityl)decyloxy-phosphonic Acid

A solution of three equivalents of imidazole in dry acetonitrile istreated dropwise with one equivalent of PCl3 over a period of 30minutes. The resulting solution is further treated with excesstriethylamine to drive the reaction to completion. After 1 hr themixture is treated with a solution of one equivalent of10-O-(dimethoxytrityl)decan-1,10-diol in dry acetonitrile and themixture stirred at room temperature for an additional hour. This mixtureis treated with an excess of a solution of triethylammonium bicarbonate,pH 8, to yield the title compound. The compound is purified by repeatedextraction of the bicarbonate solution with ethyl acetate. Pooling anddrying of the extracts over sodium bicarbonate followed by evaporationof the solvent under reduced pressure yields a compound which is used assuch without further purification. Characterization by ³¹P NMR (doublet,6 ppm, JP-H=600 Hz) and ¹H NMR yields signals for the DMT and the decanegroups as for 10-O-(dimethoxytrityl)decandiol and signals for thetriethylammonium groups (doublet, triplet, 3.2-2.2 ppm).

EXAMPLE 29 Synthesis of 10-O-(Dimethoxytrityl)decylsuccinate Half Ester

A solution of 10-O-(dimethoxytrityl)decan-1,10-diol in drydichloromethane is treated with one equivalent of succinic anhydride,excess triethylamine and 5 mole % of 4-dimethylaminopyridine catalyst.The mixture is stirred overnight under anhydrous conditions and thenfurther diluted with dichloromethane. This solution is washed with cold,saturated sodium bicarbonate, water and brine. The solution is thendried over magnesium sulfate, filtered and evaporated to dryness underreduced pressure. The resulting solid is purified by silica gel flashcolumn chromatography using ethyl acetate-methanol-triethylamine toyield the title compound as the triethylammonium salt. The free acid isobtained by repeated coevaporation of this material with wet methanol.Characterization by ¹H NMR yields signals for the DMT and decylenegroups as for 10-O-(dimethoxytrityl)decan-1,10-diol and signals for thesuccinic group (two closely spaced doublet of doublets, 2.5-3.0 ppm).

EXAMPLE 30 Derivatization of LCAA CPG with10-O-(Dimethoxytrityl)decylsuccinate Half Ester

A commercially obtained sample of controlled pore glass derivatized withlong chain alkylamine groups (LCAA CPG) is suspended in dryacetonitrile. In a separate dry container10-O-(dimethoxytrityl)decylsuccinate half ester is treated with twoequivalents of pentafluorophenol, excess triethylamine and twoequivalents of dicyclohexyl arbodiimide. The mixture containingactivated 10-O-(dimethoxytrityl)decylsuccinate half ester is stirredunder argon for one hour and then added to the suspension of CPG whilemaintaining anhydrous conditions. The mixture is then shaken gently for6 hr, the supernatant is separated and the process is repeated twicemore. The quantity of 10-O-(dimethoxytrityl)decylsuccinate half esterwhich is used in each treatment is based on the concentration ofavailable amine groups per gram of LCAA CPG, generally found to be 25-40mmoles/gram. The CPG is then treated with a dilute solution of aceticanhydride in pyridine for 1 hr to cap all unreacted aminefunctionalities and then washed several times with acetonitrile. Theextent to which this CPG has been derivatized is determined by treatingan accurately weighed sample of the resulting CPG with 2% dichloroaceticacid in acetonitrile and measuring the absorbance of an aliquot of thesupernatant at 498 nm.

EXAMPLE 31 2-N-Fmoc-2-Amino-1,3-Propanediol

2-Aminol-1,3-propandiol (3.48 g, 38.2 mmol) and NaHCO₃ (8.00 g, 95.2mmol) are suspended in 150 ml H₂O/Dioxane (1:1). Fluorenylmethylchloroformate (11.4 g, 44.0 mmol) in 25 ml toluene is added dropwise.The temperature of the reaction is maintained below 25° C. during theaddition. The mixture is stirred vigorously overnight, and then quenchedwith 50 ml saturated NaHCO₃ solution and 50 ml water. The solution isextracted with 100 ml diethyl ether. The aqueous layer is acidified topH 1 with concentrated HCl, and extracted twice with ethyl acetate, andthe organic extracts are washed with brine. The solution is dried withMgSO₄, filtered and the solvent removed in vacuo. The crude material ispurified by silica gel column chromatography to give the title compound.

EXAMPLE 32 1-O-Dimethoxytrityl-N-Fmoc-2-Amino-1,3-Propanediol

A solution of N-Fmoc-2-amino-1,3-propandiol (13.79 g, 44 mmol) in drypyridine (250 ml) is cooled to 0° C. in an ice bath. Excesstriethylamine (7 ml) and 4-dimethylaminopyridine catalyst (120 mg, 1mmol) is added followed by the slow addition of dimethoxytrityl chloride(14.8 g, 44 mmol) over 30 minutes. The mixture is stirred at 0° C. untilcomplete. The resulting solution is quenched with methanol andevaporated to dryness under reduced pressure. The residue is dissolvedin saturated NaHCO₃ and extracted with EtOAc. The EtOAc extracts arewashed with cold saturated sodium bicarbonate and brine. The organicphase is separated, dried over sodium sulfate, filtered and. evaporatedunder reduced pressure. The resulting residue is purified by flashcolumn chromatography on silica gel to give the title compound.

EXAMPLE 33 1-O-Dimethoxytrityl-N-Fmoc-2-Amino-3-O-phosphonicAcid-1,3-Propanediol

A solution of imidazole (4.29 g, 63 mmol) in dry acetonitrile at 0° C.(300 ml) is treated dropwise with PCl₃ (1.77 ml, 20.3 mmol) over aperiod of 30 minutes. The resulting solution is further treated withtriethylamine (9.06 ml, 65 mmol). To the thick slurry was added1-O-dimethoxytrityl-N-Fmoc-2-amino-1,3-propanediol (3.58 g, 5.81 mmol)in anhydrous acetonitrile (150 ml) slowly over a period of 30 minutes.The mixture is allowed to warm to room temperature and stirred for 15minutes. The mixture is quenched with pyridine/water 9:1 (100 mL) andthe mixture is evaporated in vacuo to a minimum volume and extractedwith dichloromethane (2×150 ml). The dichloromethane extracts are washedwith water and evaporated in vacuo. The residue is purified by silicagel column chromatography using dichloromethane/MeOH/pyridine to givethe title compound.

EXAMPLE 34 1-O-Dimethoxytrityl-N-Fmoc-2-Amino-1,3-Propanediol SuccinateHalf Ester

1-O-Dimethoxytrityl-N-Fmoc-2-amino-1,3-propanediol is treated withsuccinic anhydride as per the general procedure of Example 60 to givethe title compound.

EXAMPLE 35

Derivatization of LCAA CPG With1-O-Dimethoxytrityl-N-Fmoc-2-Amino-1,3-Propanediol Succinate Half Ester

1-O-Dimethoxytrityl-N-Fmoc-2-amino-1,3-propanediol succinate half esteris derivatized onto LCAA CPG as per the general procedure of Example 61to give the derivatized resin.

EXAMPLE 36 1-O-Dimethoxytrityl-2-Amino-1,3-Propanediol SuccinateDerivatized Resin

The Fmoc protecting group on the 2-amino group of the1-O-dimethoxytrityl-N-Fmoc-2-amino-1,3-propanediol succinate derivatizedCPG is removed by treatment with piperidine in dimethylformamide (DMF).CPG bound 1-O-dimethoxytrityl-N-Fmoc-2-amino-1,3-propanediol is treatedwith 2 equivalents of piperidine in DMF. The CPG is then washed withacetonitril/pyridine 1:1 and then treated a second time with 2equivalents of piperidine in DMF. Finally, the CPG is washed withacetonitrile-pyridine and then acetonitrile to give the deprotectedmaterial.

EXAMPLE 37 1-O-Dimethoxytrityl-2-N-(acetylthymine)amino-1,3-PropanediolSuccinate Derivatized Resin

Method A

The 1-O-dimethoxytrityl-2-amino-1,3-propanediol succinate derivatizedresin (2.0 g, 1.0 mmol/gm loading, 1% cross-linked) is swollen indichloroethane (200 mL) and to this is added 1-carboxymethyl thymine(2.0 g, 10 mmol), [O-(7-azabenzotriazol-1-yl)-1,1,3,-tetramethyluroniumhexafluorophosphate (3.8 g, 10 mmol) and triethylamine (2.0 g, 20 mmol).The reaction mixture is heated to 40° C. for 18 hours, then cooled andthe resin is washed 5 times with dichoromethane (50 mL), then 3 timeswith diethyl ether (100 mL), and is dried at low vacuum at 40° C. for 18hours. The free flowing resin powder is used as is.

Method B

The 1-O-dimethoxytrityl-2-amino-1,3-propanediol succinate atederivatized resin (2.0 g, 1.0 mmol/gm loading, 1% cross-linked) isswollen in dichloroethane (200 mL) and to this is added HOBt (0.1 M),PyBOP (0.1 M), N-methylmorpholine (0.15 M), as solutions in DMF followedby 1-carboxymethyl thymine (2.0 g, 10 mmol). Coupling is allowed toproceed for 2-3 hours or overnight. The resin is washed 5 times withdichoromethane (50 mL), then 3 times with diethyl ether (100 mL), and isdried at low vacuum at 40° C. for 18 hours. The free flowing resinpowder is used as is.

EXAMPLE 38 Sequential Addition and Functionalization of n BackboneSegments

The dimethoxytrityl protecting group of the derivatized resin of Example37 is removed by a treatment with a solution of trichloroacetic acid (3%w/v) in dichloromethane. The solution is passed over the solid supportuntil the DMT cation color is completely gone. The solid support iswashed with dichloromethane until no trace of acid remains. The resin isthen washed with acetonitrile-pyridine (4:1) followed by a simultaneoustreatment of the CPG with 10 equivalents of1-O-dimethoxytrityl-N-Fmoc-2-amino-3-O-phosphonic acid-1,3-propanedioland 30 equivalents of adamantane carbonyl chloride inacetonitrile-pyridine. The mixture is agitated by circulating thereagents in the synthesis vessel for 2 minutes. The CPG is then brieflywashed with acetonitrile-pyridine and then treated with diisopropylphosphite adamantane carbonyl chloride to cap all unreacted hydroxylgroups. The CPG is washed with acetonitrile-pyridine and thenacetonitrile. The resulting phosphonic acid diester is reacted with alarge molar excess of diethyl amine (the amine letter) in carbontetrachloride/pyridine. The solid support is shaken for 15 minutes andthe supernatant is removed by filtration. The solid support is washedwith pyridine. A second treatment with a large molar excess of diethylamine in carbon tetrachloride/pyridine followed by shaking will ensureefficient oxidation to the phosphoramidate. The Fmoc protecting group isremoved as per the general procedure of Example 62. The resulting freeamine group is treated with 1-carboxymethyl thymine as per the procedureof Example 37. This procedure is repeated twice to give a two mer havingacetyl thymine groups corresponding to the letter and the tethercovalently bound to the amine group attached to carbon in the backbonesegment. The functional groups bound to the phosphoramidate nitrogen areethyl groups. This procedure when repeated n times will give a fullyfunctionalized oligomer that is n+1 backbone segments long.

Upon completion of the addition of the last of the desired length andconfiguration of oligomeric sequence, the solid support is washed withpyridine/acetonitrile and the phosphoramidate is cleaved from the resinby treatment with concentrated ammonium hydroxide at room temperaturefor 3 hours. Evaporation of the supernatant and purification of thephosphoramidate on an RP-18 HPLC column yields the final oligomer.

EXAMPLE 39 N-Fmoc-Aspartic Acid-β-Benzyl Ester

Aspartic acid-β-benzyl ester (150 mmol) and diisopropyl-ethylamine (66.3ml, 49.1 g, 380 mmol) are suspended in 150 ml H₂O+300 ml dioxane.Fluorenylmethyl chloroformate (43.25 g, 1.1 eq) in 100 ml dioxane isadded dropwise. The temperature of the reaction is not allowed to riseabove 10° C. during the addition. The mixture is stirred vigorouslyovernight, and most of the solvent removed in vacuo. Water and satdbicarbonate solution are added (250 ml each), and the solution extractedwith 250 ml diethyl ether, which is discarded. The aqueous layer isacidified to pH 1 with conc HCl, and extracted twice with ethyl acetate(2×300 ml), and the organic extracts washed with brine. The solution isdried with MgSO₄, filtered and the solvent removed in vacuo to give thetitle compound.

EXAMPLE 40 4-Hydroxy-2-N-Fmoc-aminobutanoic Acid

2-N-Fmoc-aminoaspartic acid-β-benzyl ester (10 mmol) is dissolved in dryTHF (100 ml), cooled to 0° C. and Lithium borohydride (15 mmol) added.The solution is stirred at 0° C. and then room temperature until thecomplete disappearance of the starting material. Excess ethyl acetate isthen added, and the solution is washed with 0.1M citric acid solution,brine and dried with MgSO₄. The crude material is purified by flashchromatography to give the title compound.

EXAMPLE 41 4-O-Dimethoxytrityl-2-N-Fmoc-aminobutanoic Acid

4-Hydroxy-2-N-Fmoc-aminobutanoic acid (30 mmol) is coevaporated with drypyridine (2×50 ml), redissolved in 200 ml dry pyridine, and cooled in anice bath. Dimethoxytrityl chloride (22.0 g, 65 mmol) is added inportions over 30 min, and the solution stirred at RT overnight. Water isthen added (10 ml), and the solution stirred until the trityl ester iscompletely hydrolyzed. The solvent is removed under reduced pressure.The residue is dissolved in CH₂Cl₂ (300 ml), washed with 150 ml 0.1 Mcitric acid solution, 150 ml sat NaHCO₃, brine, and dried with MgSO₄followed by evaporation. The residue is purified by flashchromatography.

EXAMPLE 42 4-O-Dimethoxytrityl-2-N-Fmoc-aminobutan-1-ol

To a stirred solution of 4-O-Dimethoxytrityl-2-N-Fmoc-aminobutanoic acid(140 mmol) in 500 ml THF is added Boranemethyl sulfide (290 mmol, 21.8g, 27.3 ml) dropwise at RT. Stirring is continued until the reaction iscomplete. Methanol is carefully added (vigorous H₂ evolution), and theresulting solution stirred for a further 15 min. The solvent isevaporated under reduced pressure, and the residual gum coevaporatedwith 2×300 ml MeOH. The product is purified by flash chromatography.

EXAMPLE 43 1-O-Dimethoxytrityl-2-N-Fmoc-2-Amino-4-PhosphonicAcid-1,4-Butanediol

1-O-Dimethoxytrityl-2-N-Fmoc-2-aminobutan-1-ol is treated as per thegeneral procedure of Example 59 to give the title compound.

EXAMPLE 44 1-O-Dimethoxytrityl-2-N-Fmoc-2-Aminobutan-1-ol-Succinic AcidHalf Ester

1-O-Dimethoxytrityl-2-N-Fmoc-2-aminobutan-1-ol is treated as per thegeneral procedure of Example 60 to give the title compound.

EXAMPLE 45 1-Derivatization of LCAA CPG With1-O-Dimethoxytrityl-2-N-Fmoc-2-Amino-4-PhosphonicAcid-1,4-Butanediol-Succinic Acid Half Ester

1-O-Dimethoxytrityl-2-N-Fmoc-2-aminobutan-1-ol-succinic acid half esteris treated as per the general procedure of Example 61 to give thederivatized resin.

EXAMPLE 46 1-O-Dimethoxytrityl-2-Amino-4-Phosphonic Acid-1,4-ButanediolDerivatized Resin

The derivatezed resin of Example 45 is treated as per the generalprocedure of Example 62 to remove the Fmoc protecting group giving thetitle compound attached to resin.

EXAMPLE 47 1-O-Dimethoxytrityl-2-(2-N-Acetylthymine)-Amino-4-PhosphonicAcid-1,4-Butanediol Derivatized Resin

The derivatized resin of Example 46 is treated with N-1-thymine-2-aceticacid as per the procedure of Example 37 to give the title compoundattached to resin.

EXAMPLE 48 Synthesis of a 3-mer Having the 2-Amino-1,4-ButanediolBackbone Segment

1-O-Dimethoxytrityl-2-(2-N-acetylthymine)-Amino-4-PhosphonicAcid-1,4-Butanediol Derivatized Resin is treated with1-O-dimethoxytrityl-2-N-Fmoc-2-amino-4-phosphonic acid-1,4-butanediol,morpholine, and N-1-thymine acetic acid as per the procedure of Example37 and Example 70 to give a 3 mer with 2-N-acetylthymine bound to theamino groups and morpholine groups as the phosphoramidate substituent.

EXAMPLE 49 4-(N-Fmoc)-Amino-Glutamic Acid-γ-methyl Ester

Glutamic acid-γ-methyl ester (150 mmol) and diisopropylethylamine (66.3ml, 49.1 g, 380 mmol) are suspended in 150 ml H₂O+300 ml dioxane.Fluorenylmethyl chlorofornate (43.25 g, 1.1 eq) in 100 ml dioxane isadded dropwise. The temperature of the reaction is not allowed to riseabove 10° C. during the addition. The mixture is stirred vigorouslyovernight, and most of the solvent removed in vacuo. Water and satdbicarbonate solution are added (250 ml each), and the solution extractedwith 250 ml diethyl ether, which is discarded. The aqueous layer isacidified to pH 1 with conc HCl, and extracted twice with ethyl acetate(2×300 ml), and the organic extracts washed with brine. The solution isdried with MgSO₄, filtered and the solvent removed in vacuo to give thetitle compound.

EXAMPLE 50 5-Hydroxy-4-N-Fmoc-aminopentanoic Acid Methyl Ester

To a solution of 4-(N-Fmoc)-amino-glutamic acid-γ-methyl ester (140mmol) in 500 ml THF is added Borane-methyl sulfide (290 mmol, 21.8 g,27.3 ml) dropwise at RT (3 neck flask, mechanical stirrer, condenser,dropping funnel). After the evolution of H₂ has ceased, the solution isheated to reflux with vigorous stirring. After 1 hr a white recipitatehas formed. Methanol is carefully added (vigorous H₂ evolution), and theresulting solution refluxed for a further 15 min. The solution is cooledto RT, the solvents evaporated under reduced pressure, and the residualgum coevaporated with 2×300 ml MeOH. The product is purified by flashchromatography.

EXAMPLE 51 5-O-Dimethoxytrityl-4-Fmoc-aminopentanoic Acid Methyl Ester

5-Hydroxy-4-Fmoc-aminopentanoic acid methyl ester (30 mmol) iscoevaporated with dry pyridine (2×50 ml), redissolved in 200 ml drypyridine, and cooled in an ice bath. Dimethoxytrityl chloride (11.0 g,32.5 mmol) is added in portions over 30 min, and the solution stirred at0° C. overnight. Methanol is then added (10 ml), and the solvent removedunder reduced pressure. The resulting gum is redissolved in toluene (100ml), filtered to remove the pyridinium hydrochloride and taken todryness again. The residue is dissolved in CH₂Cl₂ (300 ml), washed with150 ml 0.1 M citric acid solution, 150 ml sat NaHCO₃, brine, and driedwith MgSO₄ followed by evaporation. The residue is purified by flashchromatography to give the title compound.

EXAMPLE 52 5-O-Dimethoxytrityl-4-Fmoc-aminopentan-1-ol

5-O-Dimethoxytrityl-4-Fmoc-aminopentanoic acid methyl ester (10 mmol) isdissolved in dry THF (100 ml), cooled to 0° C. and Lithium borohydride(10 mmol) added. The solution is stirred at 0° C. and then roomtemperature until the complete disappearance of the starting material.Excess ethyl acetate is then added, and the solution washed with 0.1Mcitric acid solution, sat NaHCO₃, brine and dried with MgSO₄. Theproduct is purified by flash chromatography

EXAMPLE 53 5-O-Dimethoxytrityl-4-N-Fmoc-2-aminopentan-1-ol HydrogenPhosphonate

Imidazole (6.81 g, 100 mmol) is dissolved in 400 ml dry CH₃CN and cooledto 0° C. Phosphorus trichloride (2.62 ml, 4.12 g, 30 mmol) is addeddropwise, followed by triethylamine (21 ml, 15.2 g, 150 mmol). A thickslurry develops to which is added over 15 min a solution of5-O-dimethoxytrityl-4-N-Fmoc-2-amino-1,5-pentanediol (10 mmol) in 50 mlCH₃CN. Once the addition is complete, the ice bath is removed and thesolution stirred at RT for 30 min. The reaction is stopped by theaddition of 100 ml pyridine/water (9:1). The solvent is removed and theresidue extracted (3×200 ml) with CH₂Cl₂, and washed with water. Theorganic phase is dried with MgSO4 and concentrated under reducedpressure. The product is further purified by flash chromatography usinga gradient of MeOH (1-10%) in CH₂Cl₂+1% pyridine.

EXAMPLE 54 5-O-Dimethoxytrityl-4-N-Fmoc-2-Amino-1,5-pentanediol-SuccinicAcid Half Ester

5-O-Dimethoxytrityl-4-N-Fmoc-2-amino-1,5-pentanediol is treated as perthe general procedure of Example 60 to give the title compound.

EXAMPLE 55 Derivatization of LCAA CPG With1-O-Dimethoxytrityl-2-N-Fmoc-2-Amino-5-PhosphonicAcid-1,5-Pentanediol-Succinic Acid Half Ester

1-O-Dimethoxytrityl-2-N-Fmoc-2-amino-1,5-pentanediol-succinic acid halfester is treated as per the general procedure of Example 61 to give thederivatized resin.

EXAMPLE 56 1-O-Dimethoxytrityl-2-Amino-4-Phosphonic Acid-1,5-PentanediolDerivatized Resin

The derivatized resin of Example 55 is treated as per the procedure ofExample 62 to remove the Fmoc protecting group giving the title compoundattached to resin.

EXAMPLE 57 1-O-Dimethoxytrityl-2(phenylacetyl)-Amino-5-PhosphonicAcid-1,5-pentanediol Derivatized Resin

The derivatized resin of Example 56 is treated with phenyl acetic acidas per the procedure of Example 37 to give the title compound attachedto resin.

EXAMPLE 58 Synthesis of a 3-mer Having the 2-Amino-1,5-pentanediolBackbone Segment

1-O-Dimethoxytrityl-2(phenylacetyl)-Amino-5-PhosphonicAcid-1,5-pentanediol Derivatized Resin is treated with1-O-dimethoxytrityl-2-N-Fmoc-2-amino-5-phosphonic acid-1,5-pentanediol,morpholine, and phenylacetic acid as per the procedure of Example 37 andExample 70 to give a 3 mer with phenylacetyl bound to the amino groupsand morpholine groups as the phosphoramidate substituent.

EXAMPLE 59 General Procedure for Converting Aminodiol Monomer Subunitsto the H-Phosphonate MonoestersN-Fmoc-5-Dimethoxytrityloxymethylpyrrolidine-3-O-Hydrogen PhosphonateTriethylanmonium Salt

Imidazole (6.81 g, 100 mmol) was dissolved in 400 ml dry CH3CN andcooled to 0° C. Phosphorus trichloride (2.62 ml, 4.12 g, 30 mmol) wasadded dropwise, followed by triethylamine (21 ml, 15.2 g, 150 mmol). Athick slurry developed to which was added over 15 min a solution ofN-Fmoc-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (10 mmol) in 50ml CH3CN. Once the addition was complete, the ice bath was removed andthe solution stirred at RT for 30 min. The reaction was stopped by theaddition of 100 ml Pyridine and 10 ml water. The solvent was removed.and the residue coevaporated with 100 ml Pyridine, then 100 ml toluene.The residue was then dissolved in 0.05M TEAB, extracted (3×200 ml) withCH2Cl2, and the extract washed with 0.05M TEAB. The organic phase wasdried with MgSO4 and concentrated under reduced pressure. The productwas further purified by flash chromatography using a gradient of MeOH(1-10%) in CH2Cl2+0.5% TEA.

EXAMPLE 60 General Procedure for Converting Aminodiol Monomer Subunitsto the Succinate DerivativesN-Fmoc-5-Dimethoxytrityloxymethylpyrrolidine-3-O-Succinate

N-Fmoc-5-Dimethoxytrityloxymethylpyrrolidine (2.0 mmol), succinicanhydride (300 mg, 3.0 mmol), DMAP (1.0 mmol, 120 mg) and triethylamine(0.4 ml, 3.0 mmol) are dissoLved in 10 ml dichloroethane and heated to50° C. for 15 min. The solution is cooled, extracted withdichloromethane, washed with 0.1M citric acid, water, brine, dried andevaporated. The residue is filtered through a short pad of silica andused directly.

EXAMPLE 61 N-Fmoc-5-Dimethoxytrityloxymethylpyrrolidine-Controlled PoreGlass (N-FMOC-5-DMT-hp-CPG)

N-Fmoc-5-Dimethoxytrityloxymethylpyrrolidine-3-O-Succinate is dissolvedin dry dichloromethane (50 ml), DMAP added (250 mg, 2 mmol) followed byToluenediisocyanate (288 ul, 2.0 mmol). The mixture was swirled for 10min then 10 g LCAA-CPG is added followed by DIEA (2 mmol, 0,34 ml). Thesuspension is kept in the dark and agitated periodically for 6-16 h. Thesolid is filtered, washed with dichloromethane and ether, then suspendedin 80 ml pyridine+20 ml water. After 1 h, the support is filtered,washed with dry pyridine (5×), dichloromethane (3×), and suspended in 60ml dichloromethane, to which 10 ml TEA, 10 ml acetic anhydride, 3 mlN-methylimidazole are added. After 1 h, the support is filtered, washedextensively with dichloromethane and ether and dried. The CPG isanalyzed for loading by weighing a portion of CPG, dissolving in 0.1Mtoluenesulfonic acid and measuring the absorbance at 498 nm.

EXAMPLE 62 Deprotection of Backbone Segment Amino Combinatorial SiteProtecting Groups General Procedure

The solid support (1-10 umol) is washed with a 10% (v/v) solution ofpiperidine in DMF for 15 seconds, and suspended in the piperidinesolution for 15 minutes. The solvent is removed and a fresh portion ofpiperidine/DMF is added for a further 15 minutes. The solid support isthen washed with several portions of DMF to remove all traces ofpiperidine.

Using this procedure the Fmoc protected backbone segmen aminocombinatorial site of the solid support boundN-Fmoc-5-dimethoxytrityloxymethylpyrrolidine of Example 61 isdeprotected.

EXAMPLE 63 Coupling of Carboxylic Acids to Backbone Segment AminoCombinatorial Sites General procedure

Deprotected backbone segment amino combinatorial sites are treatedsimultaneously with a solution of a carboxylic acid (0.1 mmol/ml) anddiisopropylethylamine (0.25 mmol/ml) in DMF, and BOP reagent (0.15mmol/ml) in DMF. A tenfold excess of reagents are added at the 10 umolscale level. The reaction is allowed to proceed for 30 minutes and afurther ten equivalents of carboxylic acid and BOP is added. After 30minutes, the solid support is washed with DMF until all the reagents areremoved.

Using this procedure the deprotected backbone segment aminocombinatorial site of Example 62 was derivatized using carboxylic acidderivatized letters.

EXAMPLE 64 Removal of the DMT Protecting Group General Procedure

Aminodiol monomeric subunits having dimethoxytrityl protecting group aredeprotected using a solution of trichloroacetic acid (3% w/v) indichloromethane is passed over the solid support until the DMT cationcolor is completely gone. The solid support is washed withdichloromethane until no trace of acid remains.

Using this procedure the the DMT protecting group of Example 63 wasremoved.

EXAMPLE 65 Coupling of Sulfonyl Chlorides to Backbone Segment AminoCombinatorial Site General Procedure

To the free amine on solid support were added simultaneously a solutionof a sulfonyl chloride (0.1 mmol/ml) and diisopropylethylamine (0.25mmol/ml) in Pyridine/CH3CN. A tenfold excess of reagents was added atthe 10 umol scale level. The reaction was allowed to proceed for 30minutes, the solid support was washed with Pyridine/CH3CN until all thereagents were removed.

EXAMPLE 66 Coupling of Acyl Groups to Backbone Segment AminoCombinatorial Site General Procedure

To the free amine on solid support are added simultaneously a solutionof a activated acylating agent (0.1 mmol/ml) and diisopropylethylamine(0.25 mmol/ml) in Pyridine/CH₃CN. A tenfold excess of reagents are addedat the 10 umol scale level. The reaction is allowed to proceed for 30minutes, the solid support is washed with Pyridine/CH₃CN until all thereagents are removed. Groups useful for acylating the free aminecombinatorial site include acid halides, acid fluorides, acidimidazolides, acid anhydrides, sulfonyl chlorides, chloroformates,isocyanates, aldehydes (under reductive alkylation conditions),isothiocyanates.

EXAMPLE 67 Phosphoramidate Library Synthesis General Procedures

The general procedures described below outline the methods for thepreparation of phosphoramidate combinatorial libraries. Allmanipulations can be accomplished using an automated synthesizer todeliver solvents and reagents to a reaction vessel containing thereactants attached to a solid support, usually controlled pore glass(CPG) or Tentagel (TG). The design of such synthesizers and the loadingof the solid support allow the manipulations to be performed on scalesranging from 50 pmol to approximately 1 mmol. if desired. Themanipulations can also be carried out manually by using a syringe with aglass frit as the reaction vessel, by drawing the appropriate solutionsinto the syringe. The reactions described can be carried out on singlecompounds attached to the solid support, or on complex mixtures preparedby the technique of bead portioning/mixing. In brief, this techniquesinvolves the addition of unique reagents to an equal number of separateportions of solid support. Once the individual reactions are complete,the portions of solid support are combined and mixed thoroughly as aslurry in an appropriate solvent, dried and redivided into the number ofportions required by the number of different reagents in the next step.In this way, a unique reagent is added to a mixture of compounds, tocreate all possible combinations of the different reagents.

Oxidation of H-Phosphonate Diester Linkages To Form PhosphoramidateLinkages Having Letters

Method A: Incorporation of Letters In Predetermined Sequence

The solid support (e.g. LCAA CPG) is derivatized with a first monomericsubunit or other group having a terminal reactive group capable offorming a covalent bond with a primary or secondary hydroxyl. Example 26illustrates a non-aminodiol monomer subunit attached to a solid supportand Example 45 illustrates attachment of an aminodiol monomer subunit toa solid support. When an aminodiol monomer subunit is derivatized to thesolid support the amino group is using the general procedure of Example62 and coupled with a letter having an optional tether as illustrated inExamples 36, 37, 63, and 65. The hydroxyl protecting group is removed asillustrated in Example 64 and the next desired aminodiol monomer subunithaving a phosphonic acid monoester group on one of the hydroxyls iscondensed onto the terminal free hydroxyl of the derivatized solidsupport as per the procedure of Example 48. The resulting phosphonicacid diester is reacted with a large molar excess of a primary orsecondary amine to form the phosphoramidate having a letter attachedthereto. The amine letter is added in a solution of carbontetrachloride/pyridine. The solid support is shaken for 15 minutes andthe supernatant is removed by filtration. The solid support is washedwith pyridine. A second treatment with a large molar excess of the amineletter in carbon tetrachloride/pyridine followed by shaking will ensureefficient oxidation to the phosphoramidate. The amino protecting groupis removed and the the free amino is coupled to a letter as above.

The process of deblocking the hydroxyl, coupling another aminodiolmonomer subunit, oxidation of the H-phosphonate diester to thephosphoramidate, deprotecting the nitrogen, and coupling a letter havingan optional tether to the free amino site, is repeated to add anotheraminodiol monomer subunit to the length of the oligomeric compound.

The oxidative incorporation of letters to form phosphoramidate linkagescan be performed all at once for a uniform substitution of letters. Forthe addition of two adjacent like letters in the oligomeric structure,the oxidation step is delayed until the backbone to support all of theseletters is synthesized and all H phosphonate sites that will bear thisletter are then oxidized simultaneously.

The above steps are repeated until all of the letter of the oligomerhave been added. All letters are predetermined in this method ofsynthesis. Upon completion of the addition of the last of the desiredlength and configuration of the oligomeric compound, the solid supportis washed with pyridine/acetonitrile and the phosphoramidate is cleavedfrom the resin by treatment with concentrated ammonium hydroxide at roomtemperature for 3 hours. Evaporation of the supernatant and purificationof the phosphoramidate on an RP-18 HPLC column yields the finaloligomer.

Method B: Incorporation of Phosphoramidate Letters in a Random Sequence

The method of oligomer synthesis as described above in Method A isrepeated to synthesize the oligomer of desired length. To randomize theamine letters on the oligomer, the method of adding a letter asdescribed in Method a above is followed except that, for randomization,the amine letters in carbon tetrachloride and a suitable cosolvent areadded as a mixture, preferably one normalized for relative reactivity.Random distribution of amine letters from this mixture of amine lettersis verified experimentally by treatment of an oligomer, which has beenpreviously treated with a mixture of amine letters and subsequentlyworked up and purified, with 10% aqueous formic acid at 50-70° C. torelease the amine letters. The actual percentages of incorporation ofthe individual amine letters is then determined by HPLC analysis of thereaction mixture. and the relative individual rates are calculated.Having once determined the relative rates, in further iteration of thesequences, the concentration of amine letters within a mixture isadjusted to reflect these rate differences.

In a variation of this method of randomization, in a five mer all sitesof which are to be randomized, the oxidation is effected simultaneously.The five mer backbone is synthesized as above and a mixture of theletters added. Upon completion of the backbone synthesis, the oxidationof amine letters is effected on all five sites as a single step.

In a further variation of this method of randomization, upon completionof the synthesis of the first backbone segment, the resin is split intofive portion and each portion is individual oxidized with one of theamine letter. The individual portions of the resin are recombined and afurther backbone segment is attached thereby extending the oligomericcompound a further unit. The resin is then again split, and theindividual portion each oxidized with one of the amine letter. Thiscycle is repeated to complete the synthesis.

Coupling of letters to the free amino positions is performed as inMethod A for a predetermined sequence or Method B for a random sequence.

Method C: Incorporation of Amine Letters in Fixed/Random Sequence

Combining methods A and B above can be used to fix certain positionswhile randomizing other positions as the oligomeric compound issynthesized. This method is further used in combination with a SURF™combinatorial strategy.

EXAMPLE 68 1-Acetyl Thymine/Benzylamine Phosphoramidate OligonerSynthesis

A solid support is derivatized with 2-O-(dimethoxytrityl)ethylsuccinatehalf ester as in Example 26. The DMT protecting group is removed usingthe standard method of Example 64.4-O-Dimethoxytrityl-2-N-Fmoc-aminobutan-1-ol is treated with PCl₃ as perExample 28 to form the phosphonic acid which is condensed onto thederivatized resin as in Example 38. The Fmoc amino protecting group isremoved as per Example 4 and (N1-thymine)-2-acetic acid (Example 7) iscoupled to the resulting free amino as per the method of Example 37. Theabove methods of examples 64, 28, 37 and 38 are repeated until six ofthe above phosphonic acid residues are incorporated. The resulting sixmer is treated using the procedures of Example 67, with a large excessof benzylamine in carbon tetrachloride/pyridine. The solid support isshaken for 15 minutes and the supernatant is removed by filtration andthen washed with pyridine. A second treatment with a large excess ofbenzylamine in carbon tetrachloride/pyridine followed by shaking willinsure efficient oxidation to the phosphoramidate. The resin is washedwith pyridine/acetonitrile and then the phosphoramidate is cleaved fromthe resin by a treatment with concentrated ammonium hydroxide at roomtemperature for 3 hours. Evaporation of the supernatant and purificationof the phosphoramidate on an RP-18 HPLC column will yield the finaloligomer. The stepwise H phosphonate coupling efficiency is determinedby measuring the absorbance of the trityl ion. The resulting six merwill have a benzylamine at each of the phosphoramidate linkages and willhave an acetylthymine group at each of the backbone segment aminocombinatorial sites.

EXAMPLE 69 Hydrogen Phosphonate Coupling General Procedure

A portion of solid support (CPG or other polymeric support e.g.TentaGel) derivatized with a DMT protected alcohol linked via asuccinate linker (1 μmol) is loaded into a DNA synthesis column, andattached to an automated DNA synthesizer programmed to perform thefollowing functions:

1) Wash with dichloromethane;

2) Treat with 3% trichloroacetic acid in dichloromethane to remove theDMT protecting group;

3) Wash with dichloromethane and CH₃CN/Pyridine (1:1);

4) Coupling: addition of alternating portions of 0.2 M Adamantoylchloride or Pivaloyl chloride in CH₃CN/Py (1:1) and 0.05 M H-phosphonatemonomer in CH₃CN/Py (1:1) for 1 min;

5) Wash with CH₃CN/Pyridine (1:1);

6) Stop and proceed to an oxidation procedure for oxidation of eachlinkage independently or repeat steps 1 through 5 to add additionalmonomer subunits prior to oxidation.

The product of this sequence of reactions is an H-phosphonate diester,which is oxidized by one of several methods including those describedbelow.

Oxidation Procedure 1: Phosphodiester

The solid support-bound H-phosphonate diester is treated (manually orautomatically) with equal volumes of solution A (0.2 M I₂ in THF) andsolution B (N-methylmorpholine/H₂O/THF 1:1:8) for 5 min, followed byequal volumes of solution A and solution C (TEA/H₂O/THF 1:1:8) for 5min, followed by washing with CH₃CN/Py (1:1).

Oxidation Procedure 2: Phosphorothioate

The solid support-bound H-phosphonate diester is treated (manually orautomatically) with a solution of S₈ in CS₂/Lutidine for 30 min. Thesolid support is then washed with CH₃CN/Py (1:1).

Oxidation Procedure 3: Phosphoramidate

The solid support-bound H-phosphonate diester is treated (manually orautomatically) with a solution of the require amine (10% V/V) inCCl₄/Pyridine 1:1 for 15-30 min. The solid support is then washed withCH₃CN/Py (1:1).

EXAMPLE 70 Combinatorial Library Synthesis Having PhosphoramidateLinkages

N-Fmoc-5-dimethoxytrityloxymethyl-3-hydroxypyrrolidine (Example 2),1-O-dimethoxytrityl-N-Fmoc-2-amino-1,3-propanediol (Example 32), and4-O-dimethoxytrityl-2-N-Fmocaminobutan-1-ol (Example 42) are treated inseparate reactions with succinic anhydride as per example 60 to give thecorresponding succinyl derivatives. In a separate set of reactions eachof the above aminodiol monomer subunits are treated with PCl₃ as perExample 28 to give the corresponding H-phosphonate monoesters. A solidsupport is divided into three equal portions and each portion is treatedwith one of the succinyl derivatized aminodiol monomer subunits as perExample 61. The solid support is combined and the amino protectinggroups are removed as per the general procedure of Example 62. The solidsupport is washed with pyridine/acetonitrile, dried, and redivided intothree equal parts.

Each portion of the solid support is treated with one of(N1-Thymine)-2-acetic acid (Example 7), (N6-benzoyl-9-adenin)-2-aceticacid (Example 11), or N-4-benzoyl-1-cytosine-2-acetic acid (Example 13)following the general procedure of Example 63. The procedure of mixing,drying and redividing the solid support is repeated.

Each portion of solid support is treated with one of the H-phosphonatemonoesters above as per the procedure of Example 30, to form theH-phosphonate diesters. The procedure of mixing, drying and redividingthe solid support is repeated. Each portion of the solid support istreated with a large molar excess of one of benzylamine,2-(2aminoethyl)-1-methylpyrrolidine, or piperonyl amine in carbontetrachloride/pyridine. The solid support is shaken for 15 minutes andthe supernatant is removed by filtration and then washed with pyridine.A second treatment with a large excess of each amine letter in carbontetrachloride/pyridine followed by shaking will insure efficientoxidation to the phosphoramidate. The solid support is combined and theamino protecting groups are removed as above. The solid support iswashed with pyridine/acetonitrile, dried, and redivided.

Each portion of the solid support is treated with one of(N1-Thymine)-2-acetic acid (Example 7), (N6-benzoyl-9-adenin)-2-aceticacid (Example 11), or N-4-benzoyl-1-cytosine-2-acetic acid (Example 13)following the general procedure of Example 63. The procedure of mixing,deblocking the hydroxyl protecting group as per Example 64, drying andredividing the solid support is repeated.

Each portion of solid support is treated with one of the H-phosphonatemonoesters above as per the procedure of Example 69 to form theH-phosphonate diesters. The procedure of mixing, drying and redividingthe solid support is repeated. Each portion of the solid support istreated with a large molar excess of one of benzylamine,2-(2-aminoethyl)-1-methylpyrrolidine, or piperonyl amine in carbontetrachloride/pyridine. The solid support is shaken for 15 minutes andthe supernatant is removed by filtration and then washed with pyridine.A second treatment with a large excess of each amine letter in carbontetrachloride/pyridine followed by shaking will insure efficientoxidation to the phosphoramidate. The solid support is combined and theamino protecting groups are removed as above. The solid support iswashed with pyridine/acetonitrile, dried, and redivided.

Each portion of the solid support is treated with one of(N1-Thymine)-2-acetic acid (Example 7), (N6-benzoyl-9-adenin)-2-aceticacid (Example 11), or N-4-benzoyl-1-cytosine-2-acetic acid (Example 13)following the general procedure of Example 63.

The resulting oligomeric compounds are cleaved from the solid support bya treatment with concentrated ammonium hydroxide at room temperature for3 hours. Evaporation of the supernatant and purification of thephosphoramidate linked oligomeric compounds on an RP-18 HPLC column willyield the final combinatorial libraries consisting of all the possibleoligomeric compounds that can be prepared using the three aminodiolmonomer subunits, the three functional groups, and the three amines.

EXAMPLE 71 Combinatorial Library Synthesis Having Phosphodiester orPhosphorothioate Linkages

A three mer is synthesized using the reagents and procedures of Example68 except that oxidation of the H-phosphonate diester linkage isaccomplished using the procedures illustrated in Example 69, Procedure 1or 2, to give either uniform phosphodiester or uniform phosphorothioatelinkages.

EXAMPLE 72 Combinatorial Library Synthesis Having Mixed Phosphodiester,Phosphorothioate, and Phosphoramidate Linkages

A three mer is synthesized using the reagents and procedures of Example68 except that oxidation of the H-phosphonate diester linkage isaccomplished by dividing the solid support into three equal portions andusing the procedures illustrated in Example 69, Procedure 1, 2, or 3 togive phosphodiester, phosphorothioate, or phosphoramidate linkages.

EXAMPLE 73 Standard Oligomer Coupling Cycle Using Standard DNA SynthesisProtocols

The oligomeric compounds of the invention are synthesized on anautomated DNA synthesizer (Applied Bio-systems model 380B) as is donewith standard oligonucleotides using standard phosphoramidate chemistrywith oxidation by iodine (see, Oligonucleotide Synthesis, A PracticalApproach, M. J. Gait., ed., Oxford University Press, New York, 1990).For phosphorothioate oligomeric compounds, the standard oxidation bottleis replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the step wise thiation of the phosphite linkages.The thiation wait step is increased to 68 sec and is followed by thecapping step. After cleavage from the CPG column and deblocking inconcentrated ammonium hydroxide at 55° C. (18 hours), the oligomericcompounds can be purified by precipitation twice out of 0.5 M NaClsolution with 2.5 volumes ethanol or by HPLC chromatography using aRP-18 column. Analytical gel electrophoresis is effected in 20%acrylamide, 8 M urea, 454 mM Tris-borate buffer, pH=7.0. Phosphodiesterand phosphorothioate oligomeric compounds are judged from polyacrylamidegel electrophoresis as to material length.

EXAMPLE 74 Synthesis of Sequence Specific Pyrrolidine Oligomer HavingPhosphodiester Linkages

“Aforvirsen” is an anti-papilloma agent having the nucleobase sequence:

TTG CTT CCA TCT TCC TCG TC SEQ. ID. NO.1.

A pyrrolidine phosphodiester linked oligomer of this preselectedsequence is prepared using the T, A, C and G reagents from Examples 7,11, 13 and 14, respectively, as per the procedure of Example 73 usingiodine as the oxidation reagent to give the phosphodiester linkedoligomeric compound having the “Aforvirsen” sequence.

EXAMPLE 75 Synthesis of Sequence Specific Pyrrolidine Oligomer HavingPhosphorothioate Linkages

A pyrrolidine phosphorothioate-linked oligomer of sequence TTG CTT CCATCT TCC TCG TC SEQ. ID. NO.1 is prepared using the T, A, C and Greagents from Examples 7, 11, 13 and 14, respectively, as per theprocedure of Example 73 using 3H-1,2-benzodithiole-3-one 1,1-dioxide asthe oxidation reagent to give the phosphorothioate linked oligomericcompound.

EXAMPLE 76 Preparation ofN-(2-[1-Tyrosinyl]-acetyl)-2-hydroxymethylpyrrolidine-4-morpholinophosphoramidate-thymidine-phosphodiester-thymidineTrimer.

A controlled pore glass resin derivatized with a 3′-5′ phosphodiesterlinked dimer of thymidine [5′-Dmt-O—T—O—P(═O)—O—T-CPG] was synthesizedin a standard manner. Two aliquots of this resin (each 24.6 mg, 47.5mmole/g resin) were separately detritylated using 3% trichloroaceticacid in dichloromethane and then sequentially washed with anhydrousacetonitrile and pyridine. Each was vigorously agitated with 1 mL of asolution of theN-(2-[1-Tyrosinyl]acetyl)-2-O-dimethoxytrityloxymethyl-pyrrolidine-3-H-phosphonatemonomer (25 mM) in adamantoyl chloride (50 mM) and pyridine for fiveminutes. The resins were then washed repeatedly with anhydrous pyridineand blown dry with argon gas. To one column was added 1 mL ofiodine/pyridine/water (2/90/8) solution and agitated vigorously; to thesecond column was added 1 mL of morpholine/carbon tetrachloride/pyridine(1/5/5) solution and both were agitated for 30 minutes. The columns weresubsequently washed with pyridine and acetonitrile and then blown drywith argon gas. Products were cleaved from the CPG by treatment with 1mL of concentrated ammonium hydroxide for 30 minutes. The ammoniasolutions were evaporated to dryness and then treated with 80% aceticacid in water for 30 minutes to remove the trityl group An aliquot ofthe solution from the first column exhibited a single peak in an HPLCanalysis (27.8 min, Vyadec C-18, 260 nm, linear gradient of ammoniumacetate (pH 7)-acetonitrile, 0-75 acetonitrile in 52 minutes)corresponding to the phosphodiester trimer. A similar analysis yielded apair of HPLC peaks (39.7 and 39.9 min) corresponding to thephosphoramidate diastereomeric trimers.

In a parallel set of experiments using the HPP-Tyrosine H-phosphonatemonomer and the 5′-Dmt-O—T—O—P(═O)—O—T-CPG the coupled resins wereoxidized to the phosphodiester trimer or to the phosphoramidate trimerusing dimethyl amine/carbon tetrachloride. These experiments yielded thephosphodiester trimer and the unresolved dimethylamine phosphoramidatetrimers at 12.4 min by an HPLC analysis, under the conditions describedbefore.

EXAMPLE 77 (5R)-O-(t-Butyldimethylsilyl)-(3R)-Hydroxypiperdine

N-Benzyl-(5R)-O-(t-butyldimethylsilyl)-(3R)-hydroxypiperidine (J. Cossy,C. Dumas, P. Michel, D. Gomez Pardo, Tetrahedron Lett., 1995, 36, 549)is dissolved in ethanol. A catalytic amount of 10% Pd/C is added, andthe solution shaken under 3 atm H₂. Once the starting material isconsumed, the catalyst is filtered and the solvent removed under reducedpressure. The product is used without further purification.

EXAMPLE 78 N-(FMOC)-(5R)-O-(t-Butyldimethylsilyl)-(3R)-Hydroxypiperidine

(5R)-O-(t-butyldimethylsilyl)-3R)-hydroxypiperidine and Na₂CO₃ (1.3 eq)are suspended in H₂O/Dioxane (1:1) 0.1M). Fluorenylmethyl chloroformate(1.1eq) in toluene is added dropwise. The temperature of the reaction isnot allowed to rise above 25° C. during the addition. The mixture isstirred vigorously overnight, acidified to pH 3 with concentrated HCl,and extracted twice with ethyl acetate. The organic extract is washedwith brine. The solution is dried with MgSO₄, filtered and the solventremoved in vacuo. The product is purified by silica gel flash columnchromatography.

EXAMPLE 79N-(FMOC)-(3R)-O-Dimethoxytrityl-(5R)-O-(t-Butyldimethylsilyl)-Piperidine

N-(FMOC)-(5R)-O-(t-butyldimethylsilyl)-(3R)hydroxypiperidine iscoevaporated with dry pyridine, and redissolved in dry pyridine (0.1M).Dimethoxytrityl chloride (1.2 eq) is added in portions over 15 minutes,and the solution stirred at RT overnight. Methanol is then added (10ml), and the solvent removed under reduced pressure. The resulting gumis redissolved in ethyl acetate, washed with 0.1 M citric acid, NaHCO₃,brine, dried with MgSO₄, and evaporated. The product is purified bysilica gel flash column chromatography.

EXAMPLE 80 N-(FMOC)-(3R)-O-Dimethoxytrityl-(5R)-Hydroxypipericline

N-(FMOC)-(3R)-O-dimethoxytrityl-(5R)-O-(t-butyldimethylsilyl)-Piperidine is dissolved in THF (0.1M), and a solution of tetrabutylammoniumfluoride (1.1 eq) in THF (1M) is added. The solution is stirred untilthe starting material is consumed. The reaction is quenched with 0.1 Mcitric acid, extracted with ethyl acetate, washed with NaHCO₃, brine anddried with MgSO₄.

EXAMPLE 81 N-(FMOC)-(3R)-O-Dimethoxytrityl-Piperidine-(5R)-O-HydrogenPhosphonate Triethylammonium Salt

The title compound is prepared fromN-(FMOC)-(3R)-O-dimethoxytrityl-(5R)-hydroxypiperidine using the generalprocedure of Example 59.

EXAMPLE 82 N-(FMOC)-(3R)-O-Dimethoxytrityl-Piperidine-(5R)-O-Succinate

The title compound is prepared fromN-(FMOC)-(3R)-O-dimethoxytrityl-(5R)-hydroxypiperidine using the generalprocedure of Example 60.

EXAMPLE 83N-(FMOC)-(3R)-O-Dimethoxytrityl-Piperidine-(5R)-O-Succinyl-CPG

The title compound is prepared fromN-(FMOC)-(3R)-O-dimethoxytrityl-piperidine-(5R)-O-succinate using thegeneral procedure of Example 61.

EXAMPLE 84 (2S)-Carboxyethyl-Pyrrolidine-5-carboxylicacid-4-methylphenylthioester

The title compound is obtained as a mixture of epimers at C5 fromN-BOC-Pyroglutamate ethyl ester using the method of J. Ezquerra, A.,Rubio, C., Pedregal, G., Sanz, J. H., Rodriguez, J. L., Garcia R.,Tetrahedron Lett. 1993, 34, 4989.

EXAMPLE 85 N-FMOC-(2S)-Carboxyethyl-Pyrrolidine-5-carboxylicacid-4-methylphenylthioester

(2S)-Carboxyethyl-pyrrolidine-5-carboxylic acid-4-methylphenylthioesterand Na₂CO₃ (1.3 eq) are suspended in H₂O/Dioxane (1:1) (0.1 M).Fluorenylmethyl chloroformate (1.1 eq) in toluene is added dropwise. Thetemperature of the reaction is not allowed to rise above 25° C. duringthe addition. The mixture is stirred vigorously overnight, acidified topH 3 with concentrated HCl, and extracted twice with ethyl acetate. Theorganic extract is washed with brine. The solution is dried with MgSO₄,filtered and the solvent removed in vacuo. The product is purified bysilica gel flash column chromatography.

EXAMPLE 86 N-FMOC-(2S)-Carboxyethyl-(5R)-Hydroxymethyl-Pyrrolidine andN-FMOC-(2S)-Carboxyethyl-(5S)-Hydroxymethyl-Pyrrolidine

The mixture of thioesters from the Example 85 are dissolved in THF andtreated with Pd(OAc) ₂ and triethylsilane. Once the thioesters arereduced to the aldehyde, the solution is cooled to 0° C., and 1 eq LiBH₄added. The reaction is stopped by the addition of acetic acid. Thesolvent is removed, and the residue extracted with ethyl acetate, washedwith 5% NaHCO3, brine and dried with MgSO4. The products (epimers) areseparated by silica gel flash column chromatography.

EXAMPLE 87N-FMOC-(2S)-Carboxyethyl-(5R)-Dimethoxytrityloxymiathyl-Pyrrolidine

N-FMOC-(2S)-carboxyethyl-(5R)-hydroxymethyl-pyrrolidin is coevaporatedwith dry pyridine, and redissolved in dry pyridine (0.1M).Dimethoxytrityl chloride (1.2 eq) is adde in portions over 15 minutes,and the solution stirred at RT overnight. Methanol is then added (10ml), and the solvent removed under reduced pressure. The resulting gumis redissolved in ethyl acetate, washed with 0.1 M citric acid, NaHCO₃,brine, dried with MgSO₄, and evaporated. The residue is purified bysilica gel flash column chromatography.

EXAMPLE 88N-FMOC-(2S)-Hydroxymethyl-(5R)-Dimethoxytrityloxymethyl-Pyrrolidine

N-FMOC-(2S)-carboxyethyl-(5R)-dimethoxytrityloxymethyl-pyrrolidine isdissolved in dry THF, cooled to 0° C., and 2 eq LiBH4 is added. Thesolution is warmed to room temperature until the starting material isconsumed. The reaction is quenched with ethyl acetate, and washed with0.1 M citric acid, NaHCO₃, brine, dried with MgSO₄ and evaporated. Theproduct is purified by silica gel flash column chromatography.

EXAMPLE 89N-FMOC-(5R)-Dimethoxytrityloxymethyl-Pyrrolidine-(2S)-oxymethyl-HydrogenPhosphonate Triethylammonium Salt

The title product is prepared fromN-FMOC-(2S)-Hydroxymethyl-(5R)-Dimethoxytrityloxymethyl-Pyrrolidineusing the general procedure of Example 59.

EXAMPLE 90 N-FMOC-(5R)-Dimethoxytrityloxymethyl-Pyrrolidine-(2S)-Oxymethyl Succinate

The title product is prepared fromN-FMOC-(2S)-Hydroxymethyl-(5R)-Dimethoxytrityloxymethyl-Pyrrolidineusing the general procedure of Example 60.

EXAMPLE 91N-FMOC-(5R)-Dimethoxytrityloxymethyl-Pyrrolidine-(2S)-Oxymethyl-Succinyl-CPG

The title product is prepared fromN-FMOC-(5R)-dimethoxytrityloxymethyl-pyrrolidine-(2S)-OxymethylSuccinate using the general procedure of Example 61.

EXAMPLE 92N-FMOC-(2S)-Carboxyethyl-(5S)-Dimethoxytrityloxymethyl-Pyrrolidine

N-FMOC-(2S)-Carboxyethyl-(5S)-Hydroxymethyl-Pyrrolidine is coevaporatedwith dry pyridine, and redissolved in dry pyridine (0.1M).Dimethoxytrityl chloride (1.2 eq) is added in portions over 15 minutesand the solution is stirred at RT overnight. Methanol (10 ml) is thenadded and the solvent removed under reduced pressure. The resulting gumis redissolved in ethyl acetate, washed with 0.1 M citric acid,concentrated NaHCO₃ solution, brine, dried with MgSO₄, and evaporated.The residue is purified by silica gel flas column chromatography.

EXAMPLE 93N-FMOC-(2S)-Hydroxymethyl-(5S)-Dimethoxytrityloxymethyl-Pyrrolidine

N-FMOC-(2S)-carboxyethyl-(5S)-dimethoxytrityloxymethyl-pyrrolidine isdissolved in dry THF, cooled to 0° C., and 2 eq LIBH₄ is added. Thesolution is warmed to room temperature until the starting material isconsumed. The reaction is quenched with ethyl acetate, and washed with0.1 M citric acid, concentrated NaHCO₃ solution, brine, dried with MgSO₄and evaporated. The product is purified by silica gel flash columnchromatography.

EXAMPLE 94N-FMOC-(5S)-Dimethoxytrityloxymethyl-Pyrrolidine-(2S)-oxymethyl-HydrogenPhosphonate Triethylammonium salt

The title product is prepared fromN-FMOC-(2S)-hydroxymethyl-(5S)-dimethoxytrityloxymethyl-pyrrolidineusing the general procedure of Example 59.

EXAMPLE 95N-FMOC-(5S)-Dimethoxytrityloxymethyl-Pyrrolidine-(2S)-OxymethylSuccinate

The title product is prepared fromN-FMOC-(2S)-Hydroxymethyl-(5S)-Dimethoxytrityloxymethyl-Pyrrolidinefusing the general procedure of Example 60.

EXAMPLE 96N-FMOC-(5S)-Dimethoxytrityloxymethyl-Pyrrolidine-(2S)-Oxymethyl-Succinyl-CPG

The title product is prepared fromN-FMOC-(5S)-Dimethoxytrityloxymethyl-Pyrrolidine-(2S)-OxymethylSuccinate using the general procedure of Example 61.

EXAMPLE 97 N-FMOC-2,2′-Dihydroxyethylamine

Diethanolamine and Na₂CO₃ (1.3 eq) are suspended in H₂O/Dioxane (1:1)(0.1M). Fluorenylmethyl chloroformate (1.1 eq) in toluene is addeddropwise. The temperature of the reaction is not allowed to rise above25° C. during the addition. The mixture is stirred vigorously overnight,acidified to pH 3 with concentrated HCl, and extracted twice with ethylacetate. The organic extract is washed with brine, dried with MgSO₄,filtered and the solvent removed in vacuo. The product is purified bysilica gel flash column chromatography.

EXAMPLE 98 N-FMOC-2′-Hydroxyethyl-2-O-Dimethoxytritylethylamine

N-FMOC-2,2′-dihydroxyethylamine is coevaporated with dr pyridine, andredissolved in dry pyridine (0.1 M). Dimethoxytrityl chloride (1.2 eq)is added in portions over 15 mininutes, and the solution stirred at RTovernight. Methanol (10 ml) is then added and the solvent removed underreduced pressure. The resulting gum is redissolved in ethyl acetate,washed with 0.1 M citric acid, concentrated NaHCO₃ solution, brine,dried with MgSO₄, and evaporated. The residue is purified by silica gelflash column chromatography.

EXAMPLE 99 N-FMOC-2-O-Dimethoxytritylethylamino-2′-O-Ethyl HydrogenPhosphonate Triethylammonium Salt

The title product is prepared fromN-FMOC-2′-Hydroxyethyl-2-O-Dimethoxytritylethylamine using the generalprocedure of Example 59.

EXAMPLE 100 N-FMOC-2,3-Propanediol

1-Aminopropanediol and Na₂CO₃ (1.3 eq) are suspended in H₂O/Dioxane(1:1) (0.1 M). Fluorenylmethyl chloroformate (1.1 eq) in toluene isadded dropwise. The temperature of the reaction is not allowed to riseabove 25° C. during the addition. The mixture is stirred vigorouslyovernight, acidified to pH 3 with concentrated HCl, and extracted twicewith ethyl acetate. The organic extract is washed with brine, dried withMgSO₄, filtered and the solvent removed in vacuo. The product ispurified by silica gel flash column chromatography.

EXAMPLE 101 N-FMOC-2-Hydroxy-3-O-Dimethoxytrityl-Propane

N-FMOC-2,3-Propanediol is coevaporated with dry pyridine, andredissolved in dry pyridine (0.1 M). Dimethoxytrityl chloride (1.2 eq)is added in portions over 15 minutes and the solution stirred at RTovernight. Methanol (10 ml)is then added and the solvent removed underreduced pressure. The resulting gum is redissolved in ethyl acetate,washed with 0.1 M citric acid, concentrated NaHCO₃ solution, brine,dried with MgSO₄, and evaporated. The residue is purified by silica gelflash column chromatography.

EXAMPLE 102 Derivatization of Backbone Segment Amino Combinatorial Siteto form Ureas General Procedure

To the free amine on solid support is added a solution of carbonyldiimidazole (0.1 mmol/ml) in DMF. A tenfold excess of reagent is addedat the 10 umol scale level. The reaction is allowed to proceed for 30min, and the reagent removed with DMF. A solution of the desired aminein DMF (10% v/v) is then added. After 30 minutes the solid support iswashed with DMF until all the reagents are removed to give thederivatized backbone segment amino combinatorial site.

EXAMPLE 103 N-FMOC-trans-3,4-bis-(Carboxymethyl)-Pyrrolidine

N-Benzyl-trans-3,4-bis-(Carboxymethyl)-Pyrrolidine is dissolved inethanol, and a catalytic amount of 10% Pd/C is added. The suspension isshaken under 3 atm H2 overnight. The catalyst is filtered, the solventremoved and the residue and Na₂CO₃ (1.3 eq) are suspended in H₂O/Dioxane(1:1) (0.1M). Fluorenylmethyl chloroformate (1.1 eq) in toluene is addeddropwise. The temperature of the reaction is not allowed to rise above25° C. during the addition. The mixture is stirred vigorously overnight,acidified to pH 3 with conc HCl, and extracted twice with ethyl acetate.The organic extract is washed with brine, dried with MgSO₄, filtered andthe solvent removed in vacuo. The product is purified by chromatography.

EXAMPLE 104 N-FMOC-trans-3,4-bis-(Hydroxymethyl)-Pyrrolidine

N-FMOC-trans-3,4-bis-(Carboxymethyl)-Pyrrolidine is dissolved in THF,and LiBH4 (4 eq) added. The solution is stirred at room temperatureuntil the esters are completely reduced. The reaction is quenched withethyl acetate and 0.1M citric acid, washed with NaHCO3 and brine, driedwith MgSO4 and evaporated. The product is purified by flashchromatography.

EXAMPLE 105N-FMOC-trans-3-(Hydroxymethyl)-4-(Dimethoxytrityloxymethyl)-Pyrrolidine

N-FMOC-trans-3,4-bis-(Hydroxymethyl)-Pyrrolidine is coevaporated withdry pyridine, and redissolved in dry pyridine (0.1M). Dimethoxytritylchloride (1.0 eq) is adder in portions over 15 min, and the solutionstirred at RT overnight. Methanol is then added (10 ml), and the solventremoved under reduced pressure. The resulting gum is redissolved inethyl acetate, washed with 0.1M citric acid, NaHCO₃, brine, dried withMgSO₄, and evaporated. The reside is purified by chromatography.

EXAMPLE 106N-FMOC-trans-3-(oxymethyl)-4-(Dimethoxytrityloxymetlhyl)-PyrrolidineHydrogen Phosphonate Triethylammonium Salt

The title product is prepared fromN-FMOC-trans-3-(Hydroxymethyl)-4-(Dimethoxytrityloxymethyl)-Pyrrolidineusing the general procedure of Example 59.

EXAMPLE 107N-FMOC-trans-3-(oxymethyl)-4-(Dimethoxytritylcoxymethyl)-PyrrolidineSuccinate

The title product is prepared fromN-FMOC-trans-3-(Hydroxymethyl)-4-(Dimethoxytrityloxymethyl)-Pyrrolidineusing the general procedure of Example 60.

EXAMPLE 108N-FMOC-trans-3-(oxymethyl)-4-(Dimethoxytrityloxymetthyl)-PyrrolidineSuccinyl CPG

The title product is prepared fromN-FMOC-trans-3-(Hydroxymethyl)-4-(Dimethoxytrityloxymethyl)-Pyrrolidineusing the general procedure of Example 61.

EXAMPLE 109 N-CBZ-cis-4-Amino-2-cyclopenten-1-ol

The product is prepared by the method of A. R. Ritter, M. J. Miller,Tetrahedron Lett. 1994, 35, 9379.

EXAMPLE 110 N-CBZ-cis-4-Amino-2,3-oxocyclopentan-1-ol

N-CBZ-cis-4-Amino-2-cyclopenten-1-ol is dissolved in dichloromethane andmeta chloroperbenzoic acid is added. The solution is stirred until thestarting material disappears. The reaction is quenched by the additionof a 5% solution of sodium bisulfite, extracted with dichloromethane,washed with NaHCO₃, brine and dried. The title compound is purified bysilica gel flash column chromatography.

EXAMPLE 111 N-CBZ-cis-4-Aminocyclopentan-1,2-diol

The epoxide of Example 110 is dissolved in THF, and treated with 2 eqLiBH₄ in the presence of a catalytic amount of Ti(OiPr)₄. Stirring iscontinued until the reaction is complete. The reaction is diluted withethyl acetate, washed with 0.1 M citric acid, NaHCO₃, brine, dried withMgSO₄ and evaporated. The title compound is purified by silica gel flashcolumn chromatography.

EXAMPLE 112 N-CBZ-cis-4-Amino-2-O-dimethoxytritylcyclopentan-1-ol

N-CBZ-cis-4-Aminocyclopentan-1,2-diol is coevaporated with dry pyridine,and redissolved in dry pyridine (0.1 M). Dimethoxytrityl chloride (1.0eq) is added in portions over 15 minutes and the solution stirred at RTovernight. Methanol is then added (10 ml), and the solvent removed underreduced pressure. The resulting gum is redissolved in ethyl acetate,washed with 0.1 M citric acid, NaHCO₃, brine dried with MgSO₄, andevaporated. The title compound is purified by silica gel flash columnchromatography.

EXAMPLE 113 N-FMOC-cis-4-Amino-2-O-dimethoxytritylcyclopentan-1-ol

N-CBZ-cis-4-Amino-2-O-dimethoxytritylcyclopentan-1-ol is dissolved inethanol and 10% Pd/C is added. The mixture is shaken under 1 atm H₂until all the material is consumed. The catalyst is filtered, thesolvent removed. The residue and Na₂CO₃ (1.3 eq) are suspended inH₂O/Dioxane (1:1) (0.1 M). Fluorenylmethyl chloroformate (1.1 eq) intoluene is added dropwise. The temperature of the reaction is notallowed to rise above 25° C. during the addition. The mixture is stirredvigorously overnight, acidified to pH 3 with concentrated HCl, andextracted twice with ethyl acetate. The organic extract is washed withbrine, dried with MgSO₄, filtered and the solvent removed in vacuo. Thetitle compound is purified by silica gel flash column chromatography.

EXAMPLE 114N-FMOC-cis-4-Amino-2-O-dimethoxytritylcyclopentan-1-O-HydrogenPhosphonate Triethylammonium Salt

The title compound is prepared using the general procedure of Example59.

EXAMPLE 115 N-CBZ-cis-4-Amino-2-cyclohexen-1-ol

The product is prepared by the method of A. R. Ritter, M. J. Miller, J.Org. Chem. 1994, 59, 4602.

EXAMPLE 116 N-CBZ-cis-4-Amino-2,3-oxocyclohexan-1-ol

N-CBZ-cis-4-Amino-2-cyclohexen-1-ol is dissolved in dichloromethane andmeta chloroperbenzoic acid was added. The solution is stirred until thestrating material disappears. The reaction is quenched by the additionof a 5% solution of sodium bisulfite, extracted with dichloromethane,washed with NaHCO₃, brine and dried. The title compound is purified bysilica gel flash column chromatography.

EXAMPLE 117 N-CBZ-cis-4-Aminocyclohexan-1,2-diol

The epoxide of the previous example is dissolved in THF and treated with2 eq LiBH₄ in the presence of a catalytic amount of Ti(OiPr)₄. Stirringis continued until the reaction is complete. The reaction is dilutedwith ethyl acetate, washed with 0.1M citric acid, NaHCO3, brine, driedwith MgSO₄ and evaporated. The title compound is purified by silica gelflash column chromatography.

EXAMPLE 118 N-CBZ-cis-4-Amino-2-O-dimethoxytritylcyclohexan-1-ol

N-CBZ-cis-4-aminocyclohexan-1,2-diol is coevaporated with dry pyridine,and redissolved in dry pyridine (0.1 M). Dimethoxytrityl chloride (1.0eq) is added in portions over 15 min, and the solution stirred at RTovernight. Methanol is then added (10 ml), and the solvent removed underreduced pressure. The resulting gum is redissolved in ethyl acetate,washed with 0.1 M citric acid, NaHCO₃, brine, dried with MgSO₄, andevaporated. The title compound is purified by silica gel flash columnchromatography.

EXAMPLE 119 N-FMOC-cis-4-Amino-2-O-dimethoxytritylcyclohexan-1-ol

N-CBZ-cis-4-Amino-2-O-dimethoxytritylcyclohexan-1-ol is dissolved inethanol and 10% Pd/C is added. The mixture is shaken under 1 atm H₂until all the material is consumed. The catalyst is filtered, thesolvent removed. The residue and Na₂CO₃ (1.3 eq) are suspended inH₂O/Dioxane (1:1) (0.1 M). Fluorenylmethyl chloroformate (1.1 eq) intoluene is added dropwise. The temperature of the reaction is notallowed to rise above 25° C. during the addition. The mixture is stirredvigorously overnight, acidified to pH 3 with concentrated HCl, andextracted twice with ethyl acetate. The organic extract is washed withbrine, dried with MgSO₄, filtered and the solvent removed in vacuo. Thetitle compound is purified by silica gel flash column chromatography.

EXAMPLE 120N-FMOC-cis-4-Amino-2-O-dimethoxytritylcyclohexan-1-O-HydrogenPhosphonate Triethylammonium Salt

The title compound is prepared using the general procedure of Example59.

EXAMPLE 121 Synthesis of a 4-mer Having Backbone Segment AminoCombinatorial Backbone Segment Amino Combinatorial SitesCombinatorialized with the Aldehydes Benzaldehyde, Aldrich-B133-4;m-tolualdehyde, Aldrich-T3,550-5; m-anisaldehyde, Aldrich-12,965-8; and3-nitrobenzaldehyde, Aldrich-N1,084-5

Method 1: Bead Splitting

A solid support is derivatized with 2-O-(dimethoxytrityl)ethylsuccinatehalf ester as in Example 26. The DMT protecting group is removed usingthe standard method of Example 64.4-O-Dimethoxytrityl-2-N-Fmoc-aminobutan-1-ol is treated with PCl₃ as perExample 28 to form the phosphonic acid which is condensed onto thederivatized resin as in Example 38. The Fmoc amino protecting group isremoved as per Example 4 and the solid support is divided into 4 equalportions and each portion is reacted with one of benzaldehyde,m-tolualdehyde, m-anisaldehyde, or 3-nitrobenzaldehyde to effectcoupling to the resulting free amino as per the method of Example 66 andthe method of Look, G. C., et.al., Tetrahedron Lett., 1995, 36,2937-2940, and the portions of solid support recombined. The combinedsolid support is treated with a solution of NaCNBH₃ or LiBH₄ intetrahydrofuran for 30 minutes followed by washing of the solid supportby methanol. The above methods are repeated until four aminodiol monomersubunits are incorporated having an equal molar mixture of each aldehydeletter at each of the amino sites. The resulting four mer is treatedusing the procedures of Example 67, with a large excess of benzylaminein carbon tetrachloride/pyridine. The solid support is shaken for 15minutes and the supernatant is removed by filtration and then washedwith pyridine. A second treatment with a large excess of benzylamine incarbon tetrachloride/pyridine followed by shaking will insure efficientoxidation to the phosphoramidate. The resin is washed withpyridine/acetonitrile and then the phosphoramidate is cleaved from theresin by a treatment with concentrated ammonium hydroxide at roomtemperature for 3 hours. Evaporation of the supernatant and purificationof the phosphoramidate on an RP-18 HPLC column will yield the finaloligomer.

Method 2

A solid support is derivatized with 2-O-(dimethoxytrityl)ethylsuccinatehalf ester as in Example 26. The DMT protecting group is removed usingthe standard method of Example 64.4-O-Dimethoxytrityl-2-N-Fmoc-aminobutan-1-ol is treated with PCl₃ as perExample 28 to form the phosphonic acid which is condensed onto thederivatized resir, as in Example 38. Following this procedure the fourmer is synthesized in four iterations of the above. The Fmoc amincprotecting groups are removed as per Example 4 and is reactedconcurrently, in one pot, with benzaldehyde, m-tolualdehyde,m-anisaldehyde, and 3-nitrobenzaldehyde. To effect this concurrentreaction 50 μmol of oligomeric compound attached to solid support isreacted with 50 μmol benzaldehyde, 50 μmol m-tolualdehyde, 50 μmolm-anisaldehyde, and 50 μmol 3-nitrobenzaldehyde in triethylorthoformate. The resulting imines are reduced as per the procedure ofmethod 1 above. In this way the four amino combinatorial sites of eacholigomeric compound attached to the solid support are combinatorializedutilizing a competitive mechanism involving a mixture of aldehydes. Theresulting four mer is treated using the procedures of Example 67, with alarge excess of benzylamine in carbon tetrachloride/pyridine. The solidsupport is shaken for 15 minutes and the supernatant is removed byfiltration and then washed with pyridine. A second treatment with alarge excess of benzylamine in carbon tetrachloride/pyridine followed byshaking will insure efficient oxidation to the phosphoramidate. Theresin is washed with pyridine/acetonitrile and then the phosphoramidateis cleaved from the resin by a treatment with concentrated ammoniumhydroxide at room temperature for 3 hours. Evaporation of thesupernatant and purification of the phosphoramidate on an RP-18 HPLCcolumn will yield the final oligomer.

EXAMPLE 122 Synthesis of Combinatorial Libraries Using Various SelectedAldehydes

Using the procedure of example 121 libraries are prepared fromoligomeric compounds of the invention that are derivatized with one,two, three, four or more of the following aldehydes available fromAldrich Chemical Company, Inc., Milwaukee, Wis. The Aldrich catalognumber is given in the right hand column and the compound name is givenin the left hand column:

Aromatic aldehydes 10793-5 Phenylacetaldehyde D20425Diphenylacetaldehyde 24582-8 Hydrocinnamaldehyde 24136-9Phenylpropionaldehyde 28902-7 (+/−)-3-Phenylbutyraldehyde 28899-3Alpha-amylcinnamaldehyde 16116-0 Alpha-bromocinnamaldehyde 26813-54-Stilbenecarboxaldehyde B133-4 Benzaldehyde 11755-2 o-Tolualdehyde25069-4 Alpha.alpha.alpha-trifluoro-o-tolualdehyde F480-72-Fluorobenzaldehyde 12497-4 2-Chlorobenzaldehyde B5700-12-Bromobenzaldehyde 10962-2 o-Anisaldehyde 15372-9 2-EthoxybenzaldehydeN1080-2 2-Nitrobenzaldehyde T3550-5 m-Tolualdehyde 19687-8Alpha.alpha.alpha-trifluoro-m-tolualdehyde F500-5 3-FluorobenzaldehydeC2340-3 3-Chlorobenzaldehyde B5720-6 3-Chlorobenzaldehyde 12965-8m-Anisaldehyde 34648-9 3-(Trifluoromethoxy)-benzaldehyde 34199-13-(1,1,2,2-Tetrafluoroethoxy)-benzaldehyde H1980-8 3-HydroxybenzaldehydeN1084-5 3-Nitrobenzaldehyde 11528-2 Isophthaldehyde T3560-2p-Tolualdehyde 23363-3 4-Ethylbenzaldehyde 13517-84-Isopropylbenzaldehyde 22494-4Alpha.alpha.alpha-trifluoro-p-tolualdehyde 12837-6 4-Fluorobenzaldehyde11221-6 4-Chlorobenzaldehyde B5740-0 4-Bromobenzaldehyde A8810-7p-Anisaldehyde 17360-6 4-Ethoxybenzaldehyde 33363-84-Propoxybenzaldehyde 23808-2 4-Butoxybenzaldehyde 37060-64-(Trifluoromethoxy)-benzaldehyde 27486-0 Terephthaldehyde mono-(diethylacetal) 14408-8 4-Hydroxybenzaldehyde 22277-1 4-(Methylthio)benzaldehyde10976-2 4-(Dimethylamino)benzaldehyde D8625-64-(Dimethylamino)benzaldehyde 33851-6 4-(Dibutylamino)benzaldehyde29355-5 4-(3-Dimethylaminopropoxy)benzaldehyde 13017-64-Nitrobenzaldehyde T220-7 Terephthaldicarboxaldehyde 34252-13-Fluoro-2-methylbenzaldehyde 34649-72-Fluoro-3-(trifluoromethyl)-benzaldehyde 26514-42,3-Difluorobenzaldehyde 26515-2 2,6-Difluorobenzaldehyde 14124-02-Chloro-6-fluorobenzaldehyde D5650-0 2,6-Dichlorobenzaldehyde 25483-52,3-Dichlorobenzaldehyde D13020-6 2,3-Dimethoxybenzaldehyde 29250-82,6-Dimethoxybenzaldehyde 31980-5 3-Fluorosalicylaldehyde 12080-4o-Vanillin 18983-9 2,3-Dihydroxybenzaldehyde 10604-62-Chloro-6-nitrobenzaldehyde 16382-1 3-methoxy-2-nitrobenzaldehyde11750-1 2,6-Dinitrobenzaldehyde 15104-1 2,4-Dimethylbenzaldehyde 15106-82,5-Dimethylbenzaldehyde 37682-52-Chloro-5-(trifluoromethyl)benzaldehyde 26516-03,4-Difluorobenzaldehyde 26517-9 2,4-Difluorobenzaldehyde 26518-72,5-Difluorobenzaldehyde 30600-2 3-Chloro-4-fluorobenzaldehyde 34807-42-Chloro-4-fluorobenzaldehyde 33954-7 3-Bromo-3-fluorobenzaldehydeD5660-8 3,4-Dichlorobenzaldehyde 14675-7 2,4-Dichlorobenzaldehyde15212-9 3-Methyl-p-anisaldehyde 15558-6 3-Fluoro-p-anisaldehyde 15429-65-Bromo-o-anisaldehyde D13040-0 2,4-Dimethoxybenzaldehyde D13060-52,5-Dimethoxybenzaldehyde 14375-8 3,4-Dimethoxybenzaldehyde 25275-13-Ethoxy-4-methoxybenzaldehyde P4910-4 Piperonal 26459-81,4-Benzodioxan-6-carboxaldehyde 31691-1 4-Hydroxy-3-methylbenzaldehyde34606-3 2-Chloro-4-hydroxybenzaldehyde 25975-6 5-Chlorosalicylaldehyde13728-6 5-Bromosalicylaldehyde 14686-2 2-Hydroxy-5-methoxybenzaldehyde16069-5 2-Hydroxy-4-methoxybenzaldehyde 14368-53-Hydroxy-4-methoxybenzaldehyde V110-4 Vanillin 12809-03-Ethoxy-4-hydroxybenzaldehyde 34215-7 5-(Trifluoromethoxy)salicylaldehyde D10840-5 3,4-Dihydroxybenzaldehyde D10820-02,5-Dihydroxybenzaldehyde 16863-7 2,4-Dihydroxybenzaldehyde 22568-14-(Diethylamino) salicylaldehyde C5880-0 5-Chloro-2-nitrobenzaldehyde13903-3 2-Chloro-5-nitrobenzaldehyde C5870-34-Chloro-3-nitrobenzaldehyde 14432-0 4-Hydroxy-3-nitrobenzaldehyde15616-7 3-Hydroxy-4-nitrobenzaldehyde 27535-22-Hydroxy-5-nitrobenzaldehyde H4810-7 5-Hydroxy-2-nitrobenzaldehydeD19360-7 2,4-Nitrobenzaldehyde 29013-03,5-Bis(trifluoromethyl)benzaldehyde 29017-3 3,5-Difluorobenzaldehyde13940-8 3,5-Dichlorobenzaldehyde 36811-3 3,5-Dihydroxybenzaldehyde12269-2 3,5-Dimethoxybenzaldehyde 36810-5 3,5-DibenzyloxybenzaldehydeM680-8 Mesitaldehyde 29233-8 2,3,5-Trichlorobenzaldehyde 13061-35-Bromoveratraldehyde 13871-1 2,4,6-Trimethoxybenzaldehyde T6840-33,4,5-Trimethoxybenzaldehyde 14039-2 3,5-Dimethyl-4-hydroxybenzaldehyde35768-5 2,6-Dimethyl-4-hydroxybenzaldehyde 14040-63,5-Di-tert-butyl-4-hydroxybenzaldehyde hemihydrate 26181-53,5-Dichlorosalicylaldehyde 12213-0 3,5-Dibromosalicylaldehyde 28344-43,5-Diiodosalicylaldehyde 13060-5 5-Bromovanillin 12948-8 5-Iodovanillin13879-7 4,6-Dimethoxysalicylaldehyde 25871-7 5-Nitrovanillin S760-23,5-Dinitrosalicylaldehyde 25959-4 2,5-Dimethyl-p-anisaldehyde T6540-45-Bromo-2,4-dimethoxybenzaldehyde N2800-0 4-Nitrovanillin 27680-43,5-Dinitrosalicylaldehyde 15205-6 2,5-Dimethyl-p-anisaldehyde 29251-65-Bromo-2,4-dimethoxybenzaldehyde 15557-8 6-Bromoveratraldehyde 13215-22,4,5-Trimethoxybenzaldehyde 27960-9 6-Nitroveratraldehyde 13765-06-Nitropiperonal 27679-0 2,5-Dichloroterephthaldehyde 33066-32,3,4-Trifluorobenzaldehyde 29231-1 2,3,6-Trichlorobenzaldehyde 15201-32,3-Dimethyl-p-anisaldehyde 29627-9 2,4-Dimethoxy-3-methylbenzaldehyde15209-9 2,3,4-Trimethoxybenzaldehyde 26084-32,3,4-Trihydroxybenzaldehyde 32893-6 Tetrafluorobenzaldehyde 10374-8Pentafluorobenzaldehyde B3468-0 4-Biphenylcarboxaldehyde 19175-23-Phenoxybenzaldehyde B2700-5 3-Benzloxybenzaldehyde 19540-53-(4-Methylphenoxy)benzaldehyde 19592-83-(4-tert-Butylphenoxy)benzaldehyde 19539-13-[3-(Trifluoromethyl)phenoxy]benzaldehyde 19530-83-(4-chlorophenoxy)benzaldehyde 19590-13-(3,4-Dichlorophenoxy)benzaldehyde 19774-23-(3,5-Dichlorophenoxy)benzaldehyde 19589-83-(4-Methoxyphonoxy)benzaldehyde 21126-5 4-Phenoxybenzaldehyde 12371-44-Benzyloxybenzaldehyde 16361-9 4-Benzyloxy-3-methoxybenzaldehyde16395-3 3-Benzyloxy-4-methoxybenzaldehyde 34603-93-Methoxy-4-(4-nitrobenzyloxy)benzaldehyde D3600-33,4-Dibenzyloxybenzaldehyde N10-9 1-Naphthaldehyde N20-62-Naphthaldehyde 15134-3 2-Methoxy-1-naphthaldehyde 10324-14-Methoxy-1-naphthaldehyde H4535-3 2-Hydroxy-1-naphthaldehyde 27208-64-Dimethylamino-1-naphthaldehyde 38201-9 2,3-Naphthalendicarboxaldehyde15014-2 2-Fluorenecarboxaldehyde 27868-8 9-Anthraldehyde M2965-710-Methylanthracene-9-carboxaldehyde 15211-0 10-Chloro-9-anthraldehydeP1160-3 Phenanthrene-9-carboxaldehyde 14403-7 1-PyrenecarboxaldehydeAliphatic aldehydes 25254-9 Formaldehyde 11007-8 Acetaldehyde P5145-1Propionaldehyde 24078-8 Isobutyraldehyde T7150-1 TrimethylacetaldehydeB10328-4 Butyraldehyde M3347-6 2-Methylbutyraldehyde 11009-42-Ethylbutyraldehyde 14645-5 Isovaleraldehyde 35990-43,3-Dimethylbutyraldehyde 11013-2 Valeraldehyde 25856-32-Methylvaleraldehyde D19050-0 2,4-Dimethylvaleraldehyde 11560-6 HexanalE2910-9 2-Ethylhexanal 30355-0 3,5,5-Trimethylhexanal H212-0Heptaldehyde O560-8 Octyl aldehyde N3080-3 Nonyl aldehyde 12577-6 Decylaldehyde U220-2 Undecylic aldehyde M8675-8 2-Methylundecanal D22200-3Dodecyl aldehyde 26923-9 Tridecanal T1000-6 Tetradecy aldehyde 11022-1Acrolein 13303-5 Methacrolein 25614-5 2-Ethylacrolein 25613-72-Butylacrclein 13298-5 Crotonaldeliyde 19261-9 trans-2-Methyl-2-butenal29468-3 2-Ethyl-trans-2-butenal 30407-7 3-Methyl-2-butenal 26925-5trans-2-pentenal 29466-7 2-Methyl-2-pentenal 29097-12,2-Dimethyl-4-pentenal 13265-9 trans-2-Hexenal 25176-3 trans-2-Heptenal30796-3 2,6-Dimethyl-5-heptenal 26995-6 trans-2-Octenal 34364-1(R)-(+)-Citronellal 37375-3 (S)-(−)-Citronellal 25565-3 trans-2-Nonenal37562-4 cis-4-Decenal 36733-8 trans-4-Decenal 13227-6 Undecylenicaldehyde 24911-4 dis-9-hexadecenal 27221-3 Cyclopropanecarboxaldehyde10846-4 Cyclohexanecarboxaldehyde 10933-9 Cyclooctanecarboxaldehyde30441-7 3-Cyclohexylpropionaldehyde T1220-3 Tetrahydrobenzaldehyde21829-4 (S)-(−)-Perillaldehyde 26467-92,6,6-Trimethyl-1-cyclohexene-1-acetaldehyde 10937-15-Norbomen-2-carboxaldehyde 21824-3 (1R)-(−)-Myrtenal 37531-4Glyoxal-1,1-dimethyl acetal 21877-4 7-Methoxy-3,7-dimethyloctanal23254-8 3-Ethoxymethacrolein 27525-52,5-Dimethoxy-3-tetrahydrofurancarboxaldehyde 26918-22,2-Dimethyl-3-hydroxypropionaldehyde G480-2 DL-Glyceraldehyde G478-0D-Glyceraldehyde 21665-8 L-Glyceraldehyde 34140-13-(Methylthio)propionaldehyde 30583-9 3-(Dimethylamino)acrolein 36549-93-(Dimethylamino)-2-methyl-2-propenal 17733-4 Pyrubic aldehyde 27706-1(S)-(−)-2-(Methoxymethyl)-1- pyrrolidinecarboxaldehyde 29211-72-Methoxy-1-pyrrolidinecarboxaldehyde 29210-92-Methoxy-1-piperidinecarboxaldehyde

EXAMPLE 124 Synthesis of Libraries from Oligomeric Compounds UtilizingAryl Acid Halides. Use of Benzoyl Chloride Aldrich; 3-methylbenzoylChloride Aldrich-T3,550-5; 3-methoxybenzoyl Chloride, Aldrich-12,965-8;and 3-nitrobenzoyl Chloride Aldrich-N1,084-5, as Illustrative Letters

Preparation of combinatorial libraries as per Example 121 using benzoicacids and benzoic acid derivatives is effected in place of benzylaldehydes described above using the general procedure of Examples 63 and65.

EXAMPLE 125 Synthesis of Libraries from Oligomeric Compounds and ArylAcid Halides Using Various Selected Acid Halides

Using the procedure of example 124 libraries are prepared fromoligomeric compounds that are diveratized with one, two, three, four ormore of the following acid halides available from Aldrich ChemicalCompany, Inc., Milwaukee, Wis. The Aldrich catalog number is given inthe right hand column and the compound name is given in the left handcolumn:

10663-1 p-Toluoyl chloride 30253-8 3-Cyanobenzoyl chloride 13096-6(+/−)-2-Cloro-2-phenylacetyl chloride 26366-4 3-(Chloromethyl)benzoylchloride 27078-4 4-(Chloromethyl)benzoyl chloride 24947-54-(Trifluoromethyl)benzoyl chloride 19394-1 4-Chlorophenoxyacetylchloride 24948-3 2-(Trifluoromethyl)benzoyl chloride 19394-14-Chlorophenoxyacetyl chloride 24948-3 2-(Trifluoromethyl)benzoylchloride 10663-1 p-Toluoyl chloride 25027-9 3-(Trifluoromethyl)benzoylchloride S67828-7 2-(2,4,5-Trichlorophenoxy)acetyl chloride 12201-7o-Toluoyl chloride 40248-6 4-(Trifluoromethoxy)benzoyl chloride 37502-03-(Dichloromethyl)benzoyl chloride 12225-4 m-Toluoyl chloride 12482-64-Cyanobenzoyl chloride P1675-3 Phenylacetyl chloride S88415-42-(Phenylthio)propionyl chloride 15862-3 Phenoxyacetyl chloride 36475-4trans-4-Nitrocinnamoyl chloride 28882-9 4-Ethoxybenzoyl chloride 23024-3m-Anisoyl chloride S67595-4 2,3-Dibromo-3-phenylpropionyl chloride30101-9 Benzyloxyacetyl chloride 25470-3 o-Anisoyl chloride C8110-1Cinnamoyl chloride 31693-8 3-Methoxyphenylacetyl chloride A8847-6p-Anisoyl chloride 16519-0 Acetylsalicyloyl chloride 36569-64-Methoxyphenylacetyl chloride 24944-0 Hydrocinnamoyl chloride 26528-43,5-Bis(trifluoromethyl)benzoyl chloride 28350-94 Ethylbenzoyl chlorideS40503-5 2-Phenoxypropionyl chloride 33304-22,5-Bis(trifluoromethyl)benzoyl chloride S62043-2 p-Tolylacetyl chloride16171-3 3,5-Dimethoxybenzoyl chloride 42339-4(R)-(−)-A-Methoxy-A-(trifluoromethyl)- phenylacetyl chloride 26480-62,5-Dimethoxyphenylacetyl chloride 25804-0 3,4-Dimethoxybenzoyl chlorideT6980-9 3,4,5-Trimethoxybenzoyl chloride 26242-0 2,6-Dimethoxybenzoylchloride 13430-9 trans-2-Phenyl-1-cyclopropanecarbonyl chloride S62264-85-(Dimethylsulfamoyl)-2-methoxybenzoyl chloride 37383-42,4-Dimethoxybenzoyl chloride A1740-4 o-Acetylmandelic chloride 24945-94-Phenyl-1,2,3,4-tetrachloro-1,3-butadiene-1- carbonyl cloride 36848-2trans-3-(trifluoromethyl)cinnamoyl chloride 15712-0 4-tert-butylbenzoylchloride S42860-4 2-Phenylbutyryl chloride 22203-8 4-Butylbenzoylchloride 23747-7 3,4-Dimethoxyphenylacetyl chloride 22204-64-Butoxybenzoyl chloride S65659-3 2-(4-Chlorobenzoyl)benzoyl chloride22214-3 4-Pentylbenzoyl chloride C3928-8 2-Chloro-2,2-diphenylacetylchloride S43639-9 4(4-Nitrophenylazo)benzoyl chloride 33158-9Diphenylacetyl chloride S80926-8 4-(Phenylazo)benzoyl chloride S61661-32-Diphenylacetyl chloride 16114-4 4-Biphenylcarbonyl chloride 22209-74-Hexylbenzoyl chloride 22205-4 4-Heptyloxybenzoyl chloride 22211-94-Hexyloxybenzoyl chloride 22206-2 4-Heptyloxybenzoyl chloride

EVALUATION

PROCEDURE 1

Antimicrobial Assay

Staphylococcus aureus

Staphylococcus aureus is known to cause localized skin infections as aresult of poor hygiene, minor trauma, psoriasis or eczema. It alsocauses respiratory infections, pneumonia, toxic shock syndrome andsepticemia. It is a common cause of acute food poisoning. It exhibitsrapid emergence of drug resistance to penicillin, cephalosporin,vancomycin and nafcillin.

In this assay, the strain S. aureus ATCC 25923 (America Type CultureCollection) is used in the bioassay. To initiate the exponential phaseof bacterial growth prior to the assay, a sample of bacteria grownovernight at 37° C. in typtocase soy broth (BBL) is used to reinoculatesample wells of 96-well microtiter plates. The assays are carried out inthe 96-well microtiter plates in 150 μL volume with approximately 1×10⁶cells per well.

Bacteria in typtocase soy broth (75 μL) is added to the compoundmixtures of the invention in solution in 75μ water in the individualwell of the microtiter plate. Final concentrations of the compoundmixtures are 25 μM, 10 μM and 1 μM. Each concentration of the compoundmixtures are assayed in triplicate. The plates are incubated at 37° C.and growth monitored over a 24 hour period by measuring the opticaldensity at 595 nm using a BioRad model 3550 UV microplate reader. Thepercentage of growth relative to a well containing no compound isdetermined. Ampicillin and tetracycline antibiotic positive controls areconcurrently tested in each screening assay.

PROCEDURE 2

Antimicrobial Mechanistic Assay

Bacterial DNA Gyrase

DNA gyrase is a bacterial enzyme which can introduce negative supercoilsinto DNA utilizing the free energy derived from ATP hydrolysis. Thisactivity is critical during DNA replication and is a well characterizedtarget for antibiotic inhibition of bacterial growth. In this assay,libraries of compounds of the invintion are screened for inhibition ofDNA gyrase. The assay measures the supercoiling of a relaxed plasmid byDNA gyrase as an electrophoretic shift on an agarose gel. Initially alllibrary pools are screened for inhibitory activity at 30 μM and then adose response analysis is effected with active subsets. Novobiocin, anantibiotic that binds to the subunit of DNA gyrase is used as a positivecontrol in the assay. The sensitivity of the DNA gyrase assay wasdetermined by titrating the concentration of the know DNA gyraseinhibitor, Novobiocin, in the supercoiling assay. The IC₅₀ wasdetermined to be 8 nM, sufficient to identify the activity of a singleactive species of comparable activity in a library having 30 μMconcentration.

PROCEDURE 3

Use of a Combinatorial Library for Identifying of Metal Chelators andImaging Agents

This procedure is used to identify compounds of the invention fromlibraries of compounds constructed to include a ring that contains anultraviolet chromophore. Further the diversity groups attached to thecompound bridge are selected from metal binders, coordinating groupssuch as amine, hydroxyl and carbonyl groups, and other groups havinclone pairs of electrons, such that the oligomeric compounds of theinvention can form coordination complexes with heavy metals and imagingagents. The procedure is used to identifs oligomeric compounds of theinvention for chelating and removing heavy metals from industrialbroths, waste stream eluents, heavy metal poisoning of farm animals andother sources of contaminating heavy metals, and for use in identifyingimaging agent carriers, such as carriers for technetium 99.

An aliquot of a test solution having the desired ion or imaging agent ata known concentration is added to an aliquot of standard solution of thepool of compounds of the invention being assayed. The UV spectrum ofthis aliquot is measured and is compared to the UV spectrum of a furtheraliquot of the same solution lacking the test ion or imaging agent. Ashift in the extinction coefficient is indicative of binding of themetal ion or imaging ion to a compound in the library pool beingassayed.

PROCEDURE 4

Assay of Combinatorial Library for PLA₂ Inhibitors

A preferred target for assay of combinatorially generated pools ofcompounds is the phospholipase A₂ family. Phospholipases A₂ (PLA₂) are afamily of enzymes that hydrolyze the sn-2 ester linkage of membranephospholipids resulting in release of a free fatty acid and alysophospholipid (Dennis, E. A., The Enzymes, Vol. 16, pp. 307-353,Boyer, P. D., ed., Academic Press, New York, 1983). Elevated levels oftype II PLA₂are correlated with a number of human inflammatory diseases.The PLA₂-catalyzed reaction is the rate-limiting step in the release ofa number of pro-inflammatory mediators. Arachidonic acid, a fatty acidcommonly linked at the sn-2 position, serves as a precursor toleukotrienes, prostaglandins, lipoxins and thrcmboxanes. Thelysophospholipid can be a precursor to platelet-activating factor. PLA₂is regulated by pro-inflammatory cytokines and, thus, occupies a centralposition in the inflammatory cascade (Dennis, ibid.; Glaser et al., TiPsReviews 1992, 14, 92; and Pruzanski et al., Inflammation 1992, 16, 451).All mammalian tissues evaluated thus far have exhibited PLA₂ activity.At least three different types of PLA₂ are found in humans: pancreatic(type I), synovial fluid (type II) and cytosolic. Studies suggest thatadditional isoenzymes exist. Type I and type II, the secreted forms ofPLA₂, share strong similarity with phospholipases isolated from thevenom of snakes. The PLA₂ enzymes are important for normal functionsincluding digestion, cellular membrane remodeling and repair, and inmediation of the inflammatory response. Both cytosolic and type IIenzymes are of interest as therapeutic targets. Increased levels of thetype II PLA₂are correlated with a variety of inflammatory disordersincluding rheumatoid arthritis, osteoarthritis, inflammatory boweldisease and septic shock, suggesting that inhibitors of this enzymewould have therapeutic utility. Additional support for a role of PLA₂ inpromoting the pathophysiology observed in certain chronic inflammatorydisorders was the observation that injection of type II PLA₂ into thefootpad of rats (Vishwanath et al., Inflammation 1988, 12, 549) or intothe articular space of rabbits (Bomalaski et al., J. Tmmunol. 1991, 146,3904) produced an inflammatory response. When the protein was denaturedbefore injection, no inflammatory response was produced.

The type II PLA₂ enzyme from synovial fluid is a relatively smallmolecule (about 14 kD) and can be distinguished from type I enzymes(e.g. pancreatic) by the sequence and pattern of its disulfide bonds.Both types of enzymes require calcium for activity. The crystalstructures of secreted PLA₂ enzymes from venom and pancreatic PLA₂, withand without inhibitors, have been reported (Scott et al., Science 1990,250, 1541). Recently, the crystal structure of PLA₂ from human synovialfluid has been determined (Wery et al., Nature 1991, 352, 79). Thestructure clarifies the role of calcium and amino acid residues incatalysis. Calcium acts as a Lewis acid to activate the scissile estercarbonyl bond of 1,2-diacylglycerophospholipids and binds to the lipid,and a His-Asp side chain diad acts as a general base catalyst toactivate a water molecule nucleophile. This is consistent with theabsence of any acyl enzyme intermediates, and is also comparable to thecatalytic mechanism of serine proteases. The catalytic residues and thecalcium ion are at the end of a deep cleft (ca. 14 Å) in the enzyme. Thewalls of this cleft contact the hydrocarbon portion of the phospholipidand are composed of hydrophobic and aromatic residues. Thepositively-charged amino-terminal helix is situated above the opening ofthe hydrophobic cleft. Several lines of evidence suggest that theN-terminal portion is the interfacial binding site (Achari et al., ColdSpring Harbor Symp. Quant. Biol. 1987, 52, 441; Cho et al., J. Biol.Chem. 1988, 263, 11237; Yang et al., Biochem. J. 1989, 262, 855; andNoel et al., J. Am. Chem. Soc. 1990, 112, 3704).

Much work has been reported in recent years on the stud of the mechanismand properties of PLA₂-catalyzed hydrolysis of phospholipids. In invitro assays, PLA₂ displays a lag phase during which the enzyme adsorbsto the substrate bilayer and a process called interfacial activationoccurs. This activation may involve desolvation of the enzyme/lipidinterface or a change in the physical state of the lipid around thecleft opening. Evidence favoring this hypothesis comes from studiesrevealing that rapid changes in PLA₂ activity occur concurrently withchanges in the fluorescence of a membrane probe (Burack et al.,Biochemistry 1993, 32, 583). This suggests that lipid rearrangement isoccurring during the interfacial activation process. PLA₂ activity ismaximal around the melting temperature of the lipid, where regions ofgel and liquid-crystalline lipid coexist. This is also consistent withthe sensitivity of PLA₂ activity to temperature and to the compositionof the substrate, both of which can lead to structurally distinct lipidarrangements separated by a boundary region. Fluorescence microscopy wasused to simultaneously identify the physical state of the lipid and theposition of the enzyme during catalysis (Grainger et al., FEBS Lett.1989, 252, 73). These studies clearly show that PLA₂ binds exclusivelyat the boundary region between liquid and solid phase lipid. While thehydrolysis of the secondary ester bond of 1,2-diacylglycerophospholipidscatalyzed by the enzyme is relatively simple, the mechanistic andkinetic picture is clouded by the complexity of the enzyme-substrateinteraction. A remarkable characteristic of PLA₂ is that maximalcatalytic activity is observed on substrate that is aggregated (i.e.phospholipid above its critical micelle concentration), while low levelsof activity are observed or monomeric substrate. As a result,competitive inhibitors of PLA₂ either have a high affinity for theactive site of the enzyme before it binds to the substrate bilayer orpartitior into the membrane and compete for the active site with thephospholipid substrate. Although a number of inhibitors appear to showpromising inhibition of PLA₂ in biochemical assays (Yuan et al., J. Am.Chem. Soc. 1987, 109, 8071; Lombardo et al., J. Biol. Chem. 1985, 260,7234; Washburn et al., J. Biol. Chem. 1991, 266, 5042; Campbell et al.,J. Chem. Soc., Chem. Commun. 1988, 1560; and Davidson et al., Biochem.Biophys. Res. Commun. 1986, 137, 587), reports describing in vivoactivity are limited (Miyake et al., J. Pharmacol. Exp. Ther. 1992, 263,1302).

In one preferred embodiment, oligomeric compounds of the invention areselected for their potential to interact with and preferably inhibit,the enzyme PLA₂. Thus, compounds of the invention can be used fortopical and/or systemic treatment of inflammatory diseases includingatopic dermatitis and inflammatory bowel disease. In selecting thfunctional groups, advantage can be taken of PLA₂'s preference foranionic vesicles over zwitterionic vesicles. Preferred compounds of theinvention for assay for PLA₂ include those having aromatic diversitygroups to facilitate binding to the cleft of the PLA₂ enzyme (Oinuma etal., J. Med. Chem. 1991, 34, 2260; Marki et al., Agents Actions 1993,38, 202; and Tanaka et al., J. Antibiotics 1992, 45, 1071). Benzyl and4-hexylbenzyl groups are preferred aromatic diversity groups.PLA₂-directed oligomeric compounds of the invention can further includehydrophobic functional groups such as tetraethylene glycol groups. Sincethe PLA₂ enzyme has a hydrophobic channel, hydrophobicity is believed tobe an important property of inhibitors of the enzyme.

After each round of synthesis as described in the above examples, theresulting pools of compounds are screened for inhibition of human typeII PLA₂ enzymatic activity. The assay is effected at the conclusion ofsynthesis to identify the wining compounds of that synthesis.Concurrently, the libraries additionally can be screened in other invitro assays to determine further mechanisms of inhibition.

The pools of the oligomeric compound libraries are screened forinhibition of PLA₂ in the assay using E. coli labeled with ³H-oleic acid(Franson et al., J. Lipid Res. 1974, 15, 380; and Davidson et al., J.Biol. Chem. 1987, 262, 1698) as the substrate. Type II PLA₂ (originallyisolated from synovial fluid), expressed in a baculovirus system andpartially purified, serves as a source of the enzyme. A series ofdilutions of each the library pools is done in water: 10 μl of each poolis incubated for 5 minutes at room temperature with a mixture of 10 μlPLA₂, 20 μl 5× PLA₂ Buffer (500 mM Tris 7.0-7.5, 5 mM CaCl₂), and 50 μlwater. Samples of each pool are run in duplicate. At this point, 10 μlof ³H E. coli cells is added. This mixture is incubated at 37° C. for 15minutes. The enzymatic reaction is stopped with the addition of 50 μl 2MHCL and 50 μl fatty-acid-free BSA (20 mg/ml PBS), vortexed for 5seconds, and centrifuged at high speed for 5 minutes. 165 μl of eachsupernate is then put into a scintillation vial containing 6 ml ofscintillant (Scintiverse) and cpms are measured in a Beckman LiquidScintillation Counter. As a control, a reaction without thecombinatorial pool is run alongside the other reactions as well as abaseline reaction containing no oligomeric compounds as well as no PLA₂enzyme. CPMs are corrected for by subtracting the baseline from eachreaction data point.

Confirmation of the “winners” is made to confirm that the oligomericcompound binds to enzyme rather than substrate and that the inhibitionof any oligomeric compound selected is specific for type II PLA₂. Anassay using ¹⁴C-phosphatidyl ethanolamine (¹⁴C-PE) as substrate, ratherthan E. coli membrane, is used to insure enzyme rather than substratespecificity. Micelles of ¹⁴C-PE and deoxycholate are incubated with theenzyme and oligomer. ¹⁴C-labeled arachidonic acid released as a resultof PLA₂-catalyzed hydrolysis is separated from substrate by thin layerchromatography and the radioactive product is quantitated. The “winner”is compared to phosphatidyl ethanolamine, the preferred substrate ofhuman type II PLA₂, to confirm its activity. PLA₂ from other sources(snake venom, pancreatic, bee venom) and phospholipase C, phospholipaseD and lysophospholipase can be used to further confirm that theinhibition is specific for human type II PLA₂.

PROCEDURE 5

Probes for the Detection of Specific Proteins and mRNA in BiologicalSamples

For the reliable, rapid, simultaneous quantification of multiplevarieties of proteins or mRNA in a biological sample without the need topurify the protein or mRNA from other cellular components, a protein ormRNA of interest from a suitable biological sample, i.e., a blood bornevirus, a bacterial pathogen product in stool, urine and other likebiological samples, is identified using standard microbiologicaltechniques. A probe comprising an oligomeric compound of the inventionis identified by a combinatorial search as noted in the above examples.Preferred for the mRNA probe are compounds synthesized to include“nucleobase” diversity groups (adenine, guanine, thymine and cytosine asthe letters) complementary to at least a portion of the nucleic acidsequence of the mRNA. Preferred for the protein probe are compoundssynthesized to include chemical functional groups that act as hydrogenbond donors and acceptors, sulfhydryl groups, hydrophobic lipophilicmoieties capable of hydrophobic interactions groups and groups capableof ionic interactions. The probe is immobilized on insoluble CPG solidsupport utilizing the procedure of Pon, R. T., Protocols forOligonucleotides and Analogs, Agrawal, S., Ed., Humana Press, Totowa,N.J., 1993, p 465-496. A known aliquot of the biological sample underinvestigation is incubated with the insoluble CPG support having theprobe thereon for a time sufficient to hybridize the protein or mRNA toprobe and thus to link them via the probe to the solid support. Thisimmobilizes protein or mRNA present in the sample to the CPG support.Other non-immobilized materials and components are then washed off theCPG with a wash media suitable for use with the biological sample. ThemRNA on the support is labelled with ethidium bromide, biotin or acommercial radionucleotide and the amount of label immobilized on theCPG support is measured to indicate the amount of mRNA present in thebiological sample. In a similar a protein is also labeled andquantified.

PROCEDURE 6

Leukotriene B₄ Assay

Leukotriene B₄ (LTB₄) has been implicated in a variety of humaninflammatory diseases, and its pharmacological effects are mediated viaits interaction with specific surface cell receptors. Library subsetsare screened for competitive inhibition of radiolabeled LTB₄ binding toa receptor preparation.

A Nenquest™ Drug Discovery System Kit (NEN Research Products, Boston,Mass.) is used to select an inhibitor of the interaction of LeukotrieneB₄ (LTB₄) with receptors on a preparation of guinea pig spleen membrane.[³H] Leukotriene B₄ reagent is prepared by adding 5 mL of ligand diluent(phosphate buffer containing NaCl, MgC₁₂, EDTA and Bacitracin, pH 7.2)to 0.25 mL of the radioligand. The receptor preparation is made bythawing the concentrate, adding 35 mL of ligand diluent and swirlinggently in order to resuspend the receptor homogeneously. Reagents arekept on ice during the course of the experiment, and the remainingportions are stored at −20° C.

Library subsets prepared as per general procedure of examples above arediluted to 5 AM, 50 μM and 500 μM in phosphate buffer (1×PBS, 0.1% azideand 0.1% BSA, pH 7.2), yielding final test concentrations of 0.5 μM, 5μM and 50 μM, respectively. Samples are assayed in duplicate. [³H] LTB₄(25 μL) is added to 25 μL of either appropriately diluted standard(unlabeled LTB₄) or library subset. The receptor suspension (0.2 mL) isadded to each tube. Sample are incubated at 4° C. for 2 hours. Controlsinclude [³H] LTB₄ without receptor suspension (total count vials), andsample of ligand and receptor without library molecules (standard)

After the incubation period, the samples are filtered through GF/B paperthat had been previously rinsed with cold saline. The contents of eachtube are aspirated onto the filter paper to remove unbound ligand fromthe membrane preparation, and the tubes washed (2×4 mL) with coldsaline. The filter paper is removed from the filtration unit and thefilter disks are placed in appropriate vials for scintillation counting.Fluor is added, and the vials shaken and allowed to stand at roomtemperature for 2 to 3 hours prior to counting. The counts/minute (cpm)obtained for each sample are subtracted from those obtained from thetotal count vials to determine the net cpm for each sample. The degreeof inhibition of binding for each library subset is determined relativeto the standard (sample of ligand and receptor without librarymolecules).

Each of the published documents mentioned in this specification areherein incorporated in their entirety.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

1 1 20 DNA Artificial Sequence Description of Artificial Sequence NovelSequence 1 ttgcttccat cttcctcgtc 20

What is claimed is:
 1. An oligomeric compound consisting essentially ofat least two aminodiol monomer subunits joined by liing groups, whereineach of said aninodiol monomer subnuits has the structure I, II, or III:

wherein: n is 0 to 5; R₁ is —T—L; T is a group of formula —R—C(═O)—where R is alkyl; L is 2-thienyl or 3-thienyl; R₂ is H or an alkyl grouphaving from 1 to 10 carbons; R₃ and R₄ are each independently H, or R₃and R₄ together from an isoposhoraidate linking group.
 2. The compoundof claim 1 wherein L is 2-thienyl.
 3. The compound of claim 1 wherein Lis 3-thienyl.
 4. The compound of claim 1 wherein R is methyl.
 5. Thecompound of claim 1 wherein R₃ is H.
 6. A library of oligomers, ecah ofsaid oligomers consisting essentially of at least two aminodiol monomersubunits joined by linking groups, said aminodiol monomer subunitshaving the structure I, II or III:

wherein: n is 0 to 5; R₁ is —T—L; T is a group of formula —R—C(═O)—where R is alkyl; L is 2-thienyl or 3-thienyl; R is H or an alkyl grouphaving from 1 to 10 carbons; R₃ and R₄ are each independently H, or R₃and R₄ together form an isophoshoramidate linking group.
 7. The libraryof claim 6 wherein L is 2-thienyl.
 8. The library of claim 7 wherein Lis 3-thienyl.
 9. The library of claim 8 wherein R is methyl.
 10. Thelibrary of claim 9 wherein R₂ is H.
 11. A composition comprising acompound according to claim
 1. 12. A composition comprising a libraryaccording to claim 6.